Optically-based nanopore analysis with reduced background

ABSTRACT

The invention is directed to nanopore arrays comprising opaque layers that reduce background fluorescence in optical signal collected in applications of such arrays for analyzing molecules. In some embodiments, such arrays are used to determine characteristics of polymers, such as polynucleotides, in methods comprising the steps of translocating polymers through nanopores of such arrays wherein polymers have one or more optical labels, exciting optical labels of the polymers in a signal generation region of each nanopore extending from the opaque layer toward the direction of the excitation beam, detecting optical signals from the signal generation regions of each nanopore to determine characteristics of the polymer translocating therethrough.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/064,429, filed Jun. 20, 2018, which is a U.S. national applicationfiled under 35 U.S.C. 371 of PCT International Application No.PCT/US2017/013036 filed Jan. 11, 2017, which claims benefit of priorityto U.S. Provisional Patent Application No. 62/279,503 filed Jan. 15,2016 and 62/308,145 filed Mar. 14, 2016, each of which are incorporatedby reference herein in its entirety.

BACKGROUND

DNA sequencing technologies developed over the last decade haverevolutionized the biological sciences, e.g. van Dijk et al, Trends inGenetics, 30(9): 418-426 (2014). However, there remains a host ofchallenges that must be overcome to achieve the full potential of thetechnology, including reduction of per-run sequencing cost,simplification of sample preparation, reduction of run times, increasingsequence read lengths, improving data analysis, and the like. Singlemolecule sequencing techniques, such as nanopore-based sequencing, mayaddress some of these challenges; however, these approaches have theirown set of technical difficulties, such as, reliable nanostructurefabrication, control of DNA translocation rates, unambiguous nucleotidediscrimination, detection and processing of signals from large arrays ofnanoscale sensors, and so on, e.g. Branton et al, Nature Biotechnology,26(10): 1146-1153 (2008).

Optical detection of nucleotides has been proposed as a potentialsolution to some of the technical difficulties in the field of nanoporesequencing, for example, the difficulty of collecting independentsignals from large arrays of nanopores. However, with fluorescence-basedsignals, overcoming background noise in the optical detection of singlemolecules remains a significant challenge. This has led to the frequentuse of microscopy systems, such as total internal reflectionfluorescence (TIRF) systems, which minimize background excitation, butadded complication and expense to the detection systems.

In view of the above, it would be advantageous to nanopore sensortechnology in general and its particular applications, such as opticallybased nanopore sequencing, if methods and devices were available thataddressed the background problem of single molecule analysis with theuse of simpler and less expensive microscopy systems.

SUMMARY OF THE INVENTION

The present invention is directed to methods and devices for singlemolecule analysis using optical labels and nanopores. In one aspect,methods and devices of the invention are directed to reducing noise inoptical signals generated upon translocation of labeled polymer analytesthrough nanopores.

In some embodiments, the invention is directed to methods of determiningcharacteristics of polymers, such as polynucleotides, which comprise thefollowing steps: (a) providing a nanopore array comprising a solid phasemembrane and an opaque layer co-extensive therewith, the nanopore arraycomprising a plurality of apertures and separating a first chamber and asecond chamber, wherein each aperture provides fluid communicationbetween the first chamber and the second chamber and has a signalgeneration region and wherein the opaque layer substantially preventslight from passing through the nanopore array; (b) translocatingpolymers from the first chamber to the second chamber through theapertures, each polymer having one or more optical labels attachedthereto capable of generating an optical signal having at least a firstwavelength indicative of a characteristic of the polymer; (c) excitingwith an excitation beam having a second wavelength the optical labels ofthe polymers as they translocate through the signal generation regionsof the apertures, wherein the optical labels in the detection regionsgenerate optical signals whose first wavelength is different than thesecond wavelength; and (d) detecting optical signals from the opticallabels in the signal generation regions to determine the characteristicsof the polymers.

In some embodiments, the invention is directed to methods of determiningcharacteristics of polymers which comprise the following steps: (a)providing a nanopore array comprising a solid phase membrane having afirst side, a second side, and a plurality of apertures therethrough,wherein the solid phase membrane separates a first chamber and a secondchamber such that each aperture provides fluid communication between thefirst chamber and the second chamber and wherein the first side of thesolid phase membrane has an opaque coating thereon and each aperture hasa detection region extending from the opaque coating of the first sidetoward the second side; (b) translocating polymers from the firstchamber to the second chamber through the apertures, each polymer havingone or more optical labels attached thereto capable of generating asignal having at least a first wavelength indicative of a characteristicof the polymer; (c) illuminating from the second side of the solid phasemembrane the optical labels in the detection regions of the apertureswith an excitation beam having a second wavelength so that opticallabels in the detection regions generate signals whose first wavelengthis different than the second wavelength; (d) detecting signals from theoptical labels in the detection regions to determine the characteristicsof the polymers.

In other embodiments, the invention is directed to methods ofdetermining characteristics of polymers comprising the steps: (a)providing a nanopore array comprising a solid phase membrane having afirst side, a second side, and a plurality of apertures therethrough,wherein the solid phase membrane separates a first chamber and a secondchamber such that each aperture provides fluid communication between thefirst chamber and the second chamber and wherein the second side of thesolid phase membrane has an opaque coating thereon and each aperture hasa detection region extending from the opaque coating of the second sideinto the second chamber; (b) translocating polymers from the firstchamber to the second chamber through the apertures, each polymer havingone or more optical labels attached thereto capable of generating asignal having at least a first wavelength indicative of a characteristicof the polymer; (c) illuminating from the second side of the solid phasemembrane the optical labels in the detection regions of the apertureswith an excitation beam having a second wavelength so that opticallabels in the detection regions generate signals whose first wavelengthis different than the second wavelength; and (d) detecting signals fromthe optical labels in the detection regions to determine thecharacteristics of the polymers.

In still other embodiments, the invention is directed to methods fordetermining sequences of polynucleotides comprising the following steps:(a) providing a nanopore array comprising a solid phase membrane and anopaque layer co-extensive therewith, the nanopore array comprising aplurality of apertures and separating a first chamber and a secondchamber, wherein each aperture provides fluid communication between thefirst chamber and the second chamber and has a signal generation regionand wherein the opaque layer substantially prevents light from passingthrough the nanopore array; (b) translocating polynucleotides from thefirst chamber to the second chamber through the apertures, whereindifferent kinds of nucleotides of the polynucleotides are labeled withdifferent fluorescent labels that generate distinguishable fluorescentsignals and wherein each of said apertures constrains nucleotides of apolynucleotide to move single file through the signal generation region;(c) exciting with an excitation beam the fluorescent labels of thepolynucleotides as they translocate through the signal generationregions of the apertures; (d) detecting fluorescent signals from thefluorescent labels in the signal generation regions to determine thecharacteristics of the polymers; and (e) determining a sequence ofnucleotides from the fluorescent signals detected at the signalgeneration region of each aperture.

The present invention advantageously overcomes the problem of opticalnoise cause by direct illumination systems in optically-based nanoporeanalysis. These and other advantages of the present invention areexemplified in a number of implementations and applications, some ofwhich are summarized below and throughout the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H illustrates elements of the invention in particularembodiments.

FIGS. 2A-2B illustrate an embodiment employing a porous layer as anopaque layer.

FIG. 3 illustrates the basic components of a confocal epi-illuminationsystem.

FIGS. 4A-4C illustrate a applications of nanopore arrays with opaquelayers to methods of optically based nanopore sequencing usingquenching.

DETAILED DESCRIPTION OF THE INVENTION

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention. For example, particular nanoporetypes and numbers, particular labels, FRET pairs, detection schemes,fabrication approaches of the invention are shown for purposes ofillustration. It should be appreciated, however, that the disclosure isnot intended to be limiting in this respect, as other types ofnanopores, arrays of nanopores, and other fabrication technologies maybe utilized to implement various aspects of the systems discussedherein. Guidance for aspects of the invention is found in many availablereferences and treatises well known to those with ordinary skill in theart, including, for example, Cao, Nanostructures & Nanomaterials(Imperial College Press, 2004); Levinson, Principles of Lithography,Second Edition (SPIE Press, 2005); Doering and Nishi, Editors, Handbookof Semiconductor Manufacturing Technology, Second Edition (CRC Press,2007); Sawyer et al, Electrochemistry for Chemists, 2^(nd) edition(Wiley Interscience, 1995); Bard and Faulkner, Electrochemical Methods:Fundamentals and Applications, 2^(nd) edition (Wiley, 2000); Lakowicz,Principles of Fluorescence Spectroscopy, 3^(rd) edition (Springer,2006); Hermanson, Bioconjugate Techniques, Second Edition (AcademicPress, 2008); and the like, which relevant parts are hereby incorporatedby reference.

The present invention is directed to methods and devices foroptically-based nanopore analysis of molecules, such as nucleic acids,which comprise nanopore arrays with one or more light-blocking layers,that is, one or more opaque layers. Typically nanopore arrays arefabricated in thin sheets of material, such as, silicon, siliconnitride, silicon oxide, aluminum oxide, or the like, which readilytransmit light, particularly at the thicknesses used, e.g. less than50-100 nm. For electrical detection of analytes this is not a problem.However, in optically-based detection of labeled molecules translocatingthrough nanopores, light transmitted through an array invariably excitesmaterials outside of intended reaction sites, or signal generationregions, thereby generating optical noise, for example, from nonspecificbackground fluorescence, fluorescence from labels of molecules that havenot yet entered a nanopore, or the like. In one aspect, the inventionaddresses this problem by providing nanopore arrays with one or morelight-blocking layers that reflect and/or absorb light from anexcitation beam, thereby reducing background noise for optical signalsgenerated at intended reaction sites associated with nanopores of anarray. In some embodiments, this permits optical labels in intendedreaction sites (such as detection zones or signal generation zonesdescribed more fully below) to be excited by direct illumination. Insome embodiments, an opaque layer may be a metal layer. Such metal layermay comprise Sn, Al, V, Ti, Ni, Mo, Ta, W, Au, Ag or Cu. In someembodiments such metal layer may comprise Al, Au, Ag or Cu. In stillother embodiments, such metal layer may comprise aluminum or gold, ormay comprise solely aluminum. The thickness of an opaque layer may varywidely and depends on the physical and chemical properties of materialcomposing the layer. In some embodiments, the thickness of an opaquelayer may be at least 5 nm, or at least 10 nm, or at least 40 nm. Inother embodiments, the thickness of an opaque layer may be in the rangeof from 5-100 nm; in other embodiments, the thickness of an opaque layermay be in the range of from 10-80 nm. An opaque layer need not block(i.e. reflect or absorb) 100 percent of the light from an excitationbeam. In some embodiments, an opaque layer may block at least 10 percentof incident light from an excitation beam; in other embodiments, anopaque layer may block at least 50 percent of incident light from anexcitation beam.

FIG. 1A illustrates the above aspects of the invention for a particularembodiment. Solid state membrane (100) has opaque coating (102) on firstside (104) to form layered membrane (101) with surface (105) facingfirst chamber (112) and with second side (106) facing second chamber(114). Layered membrane (101) separates first chamber (112) from secondchamber (114) and comprises an array of apertures (110) that eachprovide fluid communication between first chamber (112) and secondchamber (114). Apertures (110) may be solid state, or synthetic,nanopores, which may be used directly, or they may be used to immobilizeprotein nanopores, through which translocation take place. Generally,apertures have diameters, or cross sectional dimensions, that are lessthan the wavelength of an excitation beam, so that light from such beamis not transmitted through the apertures. In some embodiments, apertureshave diameters of 100 nm or less. In some embodiments, the diameter of acircular aperture is 0.586 of the wavelength of the excitation beam orless.

In some embodiments of the preceding paragraph, a method of theinvention may be implemented with the following steps: (a) providing ananopore array comprising a solid phase membrane having a first side, asecond side, and a plurality of apertures therethrough, wherein thesolid phase membrane separates a first chamber and a second chamber suchthat each aperture provides fluid communication between the firstchamber and the second chamber and wherein the first side of the solidphase membrane has an opaque coating thereon and each aperture has adetection region extending from the opaque coating of the first sidetoward the second side; (b) translocating polymers from the firstchamber to the second chamber through the apertures, each polymer havingone or more optical labels attached thereto capable of generating asignal having at least a first wavelength indicative of a characteristicof the polymer; (c) illuminating from the second side of the solid phasemembrane the optical labels in the detection regions of the apertureswith an excitation beam having a second wavelength so that opticallabels in the detection regions generate signals whose first wavelengthis different than the second wavelength; and (d) detecting signals fromthe optical labels in the detection regions to determine thecharacteristics of the polymers.

In other embodiments, as exemplified in FIG. 1C, methods of theinvention may comprise the following steps: (a) providing a nanoporearray comprising a solid phase membrane having a first side, a secondside, and a plurality of apertures therethrough, wherein the solid phasemembrane separates a first chamber and a second chamber such that eachaperture provides fluid communication between the first chamber and thesecond chamber and wherein the second side of the solid phase membranehas an opaque coating thereon and each aperture has a detection regionextending from the opaque coating of the second side into the secondchamber; (b) translocating polymers from the first chamber to the secondchamber through the apertures, each polymer having one or more opticallabels attached thereto capable of generating a signal having at least afirst wavelength indicative of a characteristic of the polymer; (c)illuminating from the second side of the solid phase membrane theoptical labels in the detection regions of the apertures with anexcitation beam having a second wavelength so that optical labels in thedetection regions generate signals whose first wavelength is differentthan the second wavelength; and (d) detecting signals from the opticallabels in the detection regions to determine the characteristics of thepolymers.

In some embodiments, whenever the opaque coating or layer is a metal, anearest-neighbor nanopore distance and excitation beam wavelength areselected to minimize plasmon-mediated extraordinary transmission throughthe nanopore array. Guidance for such selections are disclosed in thefollowing references that are incorporated by reference: Ebbesen et al,Nature, 391: 667-669 (1998); Ebbesen et al, U.S. Pat. Nos. 5,973,316;6,040,936; 6,236,033; 6,856,715; 7,057,151; 7,248,756; 8,174,696; Gur etal, Optics Comm., 284: 3509-3517 (2011); Ghaemi et al, Physical ReviewB, 58: 6779-6782 (1998); Pacifici et al, Optics Express, 16(12):9222-9238 (2008); and the like. In some embodiments, a nearest-neighbornanopore distance (or expected nearest-neighbor nanopore distance, forexample, in a random (e.g. Poisson distributed) array of nanopores) isselected which approximately equals an excitation wavelength, forexample, for exciting optical labels.

In some embodiments, signal generation regions (116) (or equivalently,intended reaction sites) each include the interior of a nanopore and aregion immediately adjacent to its exit on second side (106), whereinsuch region does not overlap the equivalent regions of other nanopores.In some embodiments, labeled molecules, such as labeled nucleic acidmolecules, are loaded, or deposited, in first chamber (112) and aretranslocated through apertures of layered membrane (101) from firstchamber (112) to second chamber (114), either directly or via aninserted protein nanopores. As labeled molecules transit apertures (110)and/or exit apertures (110) they may be directly illuminated withexcitation beam (118). Excitation beam (118) may be selected orconfigured to directly excite the labeled molecules or to indirectlyexcite the labeled molecules via a FRET interaction. In some embodiments(FIG. 1B), signal generation regions (120) (or equivalently, intendedreaction sites) each include the interior of a nanopore from first side(104) to second side (106) and a region immediately adjacent to its exiton second side (106), wherein such region does not overlap theequivalent regions of other apertures (or nanopores). In still otherembodiments, signal generation regions (or intended reaction sites) eachinclude the interior of a nanopore from its entrance from a firstchamber to its exit at a second chamber and regions immediately adjacentto its entrance and its exit, wherein such regions do not overlap theequivalent regions of other apertures.

As mentioned above, apertures in layered membranes of the invention maybe employed as nanopores directly or they may be used to hold, orimmobilize, one or more protein nanopores. In some embodiments of thelatter type, one or more protein nanopores may be embedded in a lipidbilayer disposed either on a surface of the opaque layer (e.g. 105 inFIGS. 1A, 1B, or 1E) or on a surface of the second side of the layeredmembrane (e.g. 106 in FIG. 1D). In FIGS. 1D and 1E a section of alayered membrane (101) is shown with opaque layer or coating (102),solid state membrane (100), aperture (110), together with lipid bilayer(126) (which is shown on surface (106) in FIG. 1D and on surface (105)in FIG. 1E). In the particular embodiment shown, protein nanopore (122)that has label (124) attached (e.g. a donor label, such as a quantumdot, metal nanoparticle, or other fluorescent nanoparticle forgenerating FRET signals) is shown inserted into lipid bilayer (126),although as noted elsewhere, the invention comprehends protein nanoporesin such configurations without labels for FRET signal generation. Someembodiments of FIG. 1D which have a signal generation region adjacent tolabel (124) (that is, within a FRET distance of label (124)) correspondto the configuration described in FIG. 1B in which signal generationregions extend from first side (104) to a space proximal to the apertureor nanopore exit at second side (106). Similarly, some embodiments ofFIG. 1E which also have a signal generation region adjacent to label(124) (for example, within a FRET distance of label (124)) correspond tothe configuration described in FIG. 1A in which signal generationregions extend from surface (105) to a space proximal to the aperture ornanopore exits at second side (106).

In some embodiments, such as those described in FIGS. 1A-1E, the layeredmembrane may further comprise a passivation coating, such as an oxidecoating, such as a silicon oxide coating, to stabilize metal layers. Forexample, a silicon nitride membrane may be coated with an opaquematerial, such as a metal, using physical or chemical depositiontechniques to form a layered membrane, after which apertures may beetched or drilled using a focused electron or ion beam to form an arrayof apertures in the layered membrane. Such array may then be furthercoated with protecting layer, such as silicon oxide, again usingchemical or vapor deposition techniques.

In some embodiments, the invention may include a method forcharacterizing polymers, such as biological polymers including nucleicacids and proteins, by the following steps: (a) providing a nanoporearray comprising a solid phase membrane having a first side, a secondside, and a plurality of apertures therethrough, wherein the solid phasemembrane separates a first chamber and a second chamber such that eachaperture provides fluid communication between the first chamber and thesecond chamber and wherein the first side of the solid phase membranehas an opaque coating thereon and each aperture has a detection regionextending from the opaque coating of the first side toward the secondside; (b) translocating polymers from the first chamber to the secondchamber through the apertures, each polymer having one or more opticallabels attached thereto capable of generating a signal having at least afirst wavelength indicative of a characteristic of the polymer; (c)illuminating from the second side of the solid phase membrane theoptical labels in the detection regions of the apertures with anexcitation beam comprising at least a second wavelength so that opticallabels in the detection regions generate signals whose first wavelengthis different than the second wavelength; (d) detecting signals from theoptical labels in the detection regions to determine the characteristicsof the polymers. In some embodiments, polymers may be polynucleotides orproteins. In still other embodiments, polymers may be polynucleotides.In further embodiments, polynucleotides may be single stranded nucleicacids. In some embodiments, a characteristic of polymers analyzed ordetermined is a monomer sequence, such as a nucleotide sequence, of thepolymers. In some embodiments, optical labels on polymers are FRETlabels, such as described in U.S. patents and patent and internationalpublications: 8,771,491; US2013/0203050; or WO2014/190322, which areincorporated herein by reference. In some embodiments, aperturescomprise protein nanopores. Briefly, in some embodiments, a FRET labelcomprises at each signal generation region at least one FRET donor labeland at least one FRET acceptor label, wherein an excitation beam excitesthe FRET donor labels which, in turn, transfer energy to FRET acceptorlabels within a FRET distance of the donor labels which, in turn, emitan optical signal. Typically, an excitation beam comprises a secondwavelength and the optical signal comprises a first wavelength distinctfrom the second wavelength, for example, to permit use of anepi-illumination system. In some embodiments, a detection region mayextend from the opaque coating of the first side toward the second sideand include an extra-membrane space immediately proximal to the exit ofan aperture and/or nanopore. In some embodiments, such extra-membranespace does not extend beyond 50 nm from the exit of a nanopore oraperture; in other embodiments, such extra-membrane space does notextend beyond 10 nm from the exit of a nanopore or aperture.

Briefly, as described more fully in U.S. Pat. No. 8,771,491, in someembodiments, an aperture and/or nanopore may be labeled with one or moreFRET donors and polymers may each be labeled with FRET acceptors suchthat at least selected donors and acceptors form FRET pairs; that is,the emission spectra of a donor overlaps the absorption spectra of atleast one acceptor so that if other conditions are met (e.g. donorexcitation, donor and acceptor being within a FRET distance, donor andacceptor having proper relative orientation, and the like) a FRETinteraction can occur. In a FRET interaction excitation energy of thedonor is transferred to an acceptor non-radiatively, after which theacceptor, emits an optical signal that has a lower energy than theexcitation energy of the donor. Donor are usually excited byilluminating them with a light beam, such as generated by a laser.

In some embodiments, protein nanopores may be inserted in solid statemembranes without, or with only small amounts of, lipid bilayers to formarrays, as described in Huber et al, U.S. patent publication2013/0203050, which is incorporated herein by reference. Such a proteinnanopore/aperture configuration is illustrated in FIG. 1F. In thisparticular embodiment, layered membrane (101) comprising solid statemembrane (100) and opaque layer (102) comprises aperture (107) havingimmobilized therein protein nanopore (122) labeled with donor (128),which may be an optically active particle, such as a quantum dot. Asacceptor labeled polymer (130) passes through and exits bore of proteinnanopore (122), acceptor labels pass within a FRET distance of donor(128). The size of aperture (107) is fabricated to be capable ofimmobilizing a single protein nanopore. Upon direct illumination ofaperture (107) by excitation beam (133), donor (128) is excited and madecapable of entering a FRET interaction with an acceptor of labeledpolymer (130) being translocated through protein nanopore (122). On theother hand, opaque layer (102) decreases radiation from beam (133) fromilluminating acceptors on labeled polymer (131) which is located on theopposite side of opaque layer (102) as beam (133). Opaque layer (102)blocks radiation from beam (133) by either absorbing (135) and/orreflecting (137) it.

A further embodiment is illustrated in FIG. 1G in which a surface ofopaque layer (102), such as a metal layer, forms a boundary of secondchamber (114). In some embodiments, arrays of solid state apertures(146) may be fabricated using conventional micromachining techniques.For example, to a silicon substrate (140), nonconductive solid statemembrane (100), such as a silicon nitride layer, is added, for exampleby chemical vapor deposition, or like technique. In some embodiments,layer (100) is in the range of 30-100 nm. On top of layer (100), opaquelayer (102), such as a metal layer, is added, for example, by chemicalvapor deposition or like technique to form a three-layer sheet. Toopaque layer (102) a layer of photoresist may be added, which afterremoving developed photoresist material at aperture locations, holes areformed by etching through opaque layer (102) and solid state membrane(100), to form a portion of aperture (146). A second etching is carriedout through silicon layer (140) to form the rest of apertures (146) inthe array. A nanopore sensing device in accordance with one embodimentof the invention may be constructed by disposing lipid bilayer (126) onsurface (141) of opaque layer (102), which has inserted therein at leastone protein nanopore (122) with FRET donor label (124). Upon dipositionin first chamber (112) charged and labeled polymer (150), for examplecomprising acceptor labels (151) and (152) for two different kinds ofmonomers, may translocate through protein nanopore (122) whichconstrains each acceptor label (151 or 152) to come within a FRETdistance (142) of donor label (124). Donor label (124) is excited bybeam (133) and transfers excitation energy to acceptor labels withinFRET distance (142), which, in turn, emit FRET signal (144) indicativeof the monomer to which it is attached.

In still other embodiments, as illustrated in FIG. 1H, lipid bilayer(126) may be disposed on surface (104) of layer (100), so that proteinnanopore (122) is inserted on a side of layered membrane (101) oppositefrom the side to which excitation beam (133) is directed.

In some embodiments, an opaque layer may comprise an opaque porouslayer, which in some embodiments may be an opaque nanoporous layer, asillustrated in FIGS. 2A and 2B. In such embodiments, layered membrane(101) comprises solid state membrane (200) and nanoporous layer (202)which comprises an opaque material having nanometer-sized pores thatprovide indirect and/or serpentine passages across the layer toapertures (204). FIG. 2B illustrates an embodiment with labeled proteinnanopores (210) inserted in lipid bilayer (206) disposed on second side(216) of solid state membrane (200). As with the embodiments of FIGS.1A-1H, nanoporous layer (202) blocks or reduces radiation directed tosecond side (216) of solid state membrane (200) from reaching labeledpolymers or other materials on the other side of nanoporous layer (202),thereby reducing the generation of undesirable noise in optical signalsfrom detection events at the exit of protein nanopore (210).

As mentioned above, in some embodiments, an epi-illumination system, inwhich excitation beam delivery and optical signal collection occursthrough a single objective, may be used for direct illumination oflabels on a polymer analyte or donors on nanopores. The basic componentsof a confocal epi-illumination system for use with the invention isillustrated in FIG. 3. Excitation beam (302) passes through dichroic(304) and onto objective lens (306) which focuses (310) excitation beam(302) onto layered membrane (300), in which labels are excited directlyto emit an optical signal, such as a fluorescent signal, of are excitedindirectly via a FRET interaction to emit an optical signal. Suchoptical signal is collected by objective lens (306) and directed todichroic (304), which is selected so that it passes light of excitationbeam (302) but reflects light of optical signals (311). Reflectedoptical signals (311) passes through lens (314) which focuses it throughpinhole (316) and onto detector (318).

In some embodiments, a device for implementing the above method forpolymers comprising single stranded nucleic acids typically includes aset of electrodes for establishing an electric field across the layeredmembrane and nanopores. Single stranded nucleic acids are exposed tonanopores by placing them in an electrolyte in a first chamber, which isconfigured as the “cis” side of the layered membrane by placement of anegative electrode in the chamber. Upon application of an electricfield, the negatively single stranded nucleic acids are captured bynanopores and translocated to a second chamber on the other side of thelayered membrane, which is configured as the “trans” side of membrane byplacement of a positive electrode in the chamber. The speed oftranslocation depends in part on the ionic strength of the electrolytesin the first and second chambers and the applied voltage across thenanopores. In optically based detection, a translocation speed may beselected by preliminary calibration measurements, for example, usingpredetermined standards of labeled single stranded nucleic acids thatgenerate signals at different expected rates per nanopore for differentvoltages. Thus, for DNA sequencing applications, a translocation speedmay be selected based on the signal rates from such calibrationmeasurements. Consequently, from such measurements a voltage may beselected that permits, or maximizes, reliable nucleotideidentifications, for example, over an array of nanopores. In someembodiments, such calibrations may be made using nucleic acids from thesample of templates being analyzed (instead of, or in addition to,predetermined standard sequences). In some embodiments, suchcalibrations may be carried out in real time during a sequencing run andthe applied voltage may be modified in real time based on suchmeasurements, for example, to maximize the acquisition ofnucleotide-specific signals.

Nanopores and Nanopore Arrays

As discussed above, nanopores used with the invention may be solid-statenanopores, protein nanopores, or hybrid nanopores comprising proteinnanopores or organic nanotubes such as carbon or graphene nanotubes,configured in a solid-state membrane, or like framework. Importantfeatures of nanopores include constraining polymer analytes, such aspolynucleotides, so that their monomers pass through a detection zone(or signal generation region) in sequence (that is, so that nucleotidespass a detection zone one at a time, or in single file). In someembodiments, additional features of nanopores include passing singlestranded nucleic acids while not passing double stranded nucleic acids,or equivalently bulky molecules.

In some embodiments, nanopores used in connection with the methods anddevices of the invention are provided in the form of arrays, such as anarray of clusters of nanopores, which may be disposed regularly on aplanar surface. In some embodiments, clusters are each in a separateresolution limited area so that optical signals from nanopores ofdifferent clusters are distinguishable by the optical detection systememployed, but optical signals from nanopores within the same clustercannot necessarily be assigned to a specific nanopore within suchcluster by the optical detection system employed.

Solid state nanopores may be fabricated in a variety of materialsincluding but not limited to, silicon nitride (Si₃N₄), silicon dioxide(SiO₂), and the like. The fabrication and operation of nanopores foranalytical applications, such as DNA sequencing, are disclosed in thefollowing exemplary references that are incorporated by reference: Ling,U.S. Pat. No. 7,678,562; Hu et al, U.S. Pat. No. 7,397,232; Golovchenkoet al, U.S. Pat. No. 6,464,842; Chu et al, U.S. Pat. No. 5,798,042;Sauer et al, U.S. Pat. No. 7,001,792; Su et al, U.S. Pat. No. 7,744,816;Church et al, U.S. Pat. No. 5,795,782; Bayley et al, U.S. Pat. No.6,426,231; Akeson et al, U.S. Pat. No. 7,189,503; Bayley et al, U.S.Pat. No. 6,916,665; Akeson et al, U.S. Pat. No. 6,267,872; Meller et al,U.S. patent publication 2009/0029477; Howorka et al, Internationalpatent publication WO2009/007743; Brown et al, International patentpublication WO2011/067559; Meller et al, International patentpublication WO2009/020682; Polonsky et al, International patentpublication WO2008/092760; Van der Zaag et al, International patentpublication WO2010/007537; Yan et al, Nano Letters, 5(6): 1129-1134(2005); Iqbal et al, Nature Nanotechnology, 2: 243-248 (2007); Wanunu etal, Nano Letters, 7(6): 1580-1585 (2007); Dekker, Nature Nanotechnology,2: 209-215 (2007); Storm et al, Nature Materials, 2: 537-540 (2003); Wuet al, Electrophoresis, 29(13): 2754-2759 (2008); Nakane et al,Electrophoresis, 23: 2592-2601 (2002); Zhe et al, J. Micromech.Microeng., 17: 304-313 (2007); Henriquez et al, The Analyst, 129:478-482 (2004); Jagtiani et al, J. Micromech. Microeng., 16: 1530-1539(2006); Nakane et al, J. Phys. Condens. Matter, 15 R1365-R1393 (2003);DeBlois et al, Rev. Sci. Instruments, 41(7): 909-916 (1970); Clarke etal, Nature Nanotechnology, 4(4): 265-270 (2009); Bayley et al, U.S.patent publication 2003/0215881; and the like.

In some embodiments, the invention comprises nanopore arrays with one ormore light-blocking layers, that is, one or more opaque layers.Typically nanopore arrays are fabricated in thin sheets of material,such as, silicon, silicon nitride, silicon oxide, aluminum oxide, or thelike, which readily transmit light, particularly at the thicknessesused, e.g. less than 50-100 nm. For electrical detection of analytesthis is not a problem. However, in optically-based detection of labeledmolecules translocating nanopores, light transmitted through an arrayinvariably excites materials outside of intended reaction sites, thusgenerates optical noise, for example, from nonspecific backgroundfluorescence, fluorescence from labels of molecules that have not yetentered a nanopore, or the like. In one aspect, the invention addressesthis problem by providing nanopore arrays with one or morelight-blocking layers that reflect and/or absorb light from anexcitation beam, thereby reducing background noise for optical signalsgenerated at intended reaction sites associated with nanopores of anarray. In some embodiments, this permits optical labels in intendedreaction sites to be excited by direct illumination. In someembodiments, an opaque layer may be a metal layer. Such metal layer maycomprise Sn, Al, V, Ti, Ni, Mo, Ta, W, Au, Ag or Cu. In some embodimentssuch metal layer may comprise Al, Au, Ag or Cu. In still otherembodiments, such metal layer may comprise aluminum or gold, or maycomprise solely aluminum. The thickness of an opaque layer may varywidely and depends on the physical and chemical properties of materialcomposing the layer. In some embodiments, the thickness of an opaquelayer may be at least 5 nm, or at least 10 nm, or at least 40 nm. Inother embodiments, the thickness of an opaque layer may be in the rangeof from 5-100 nm; in other embodiments, the thickness of an opaque layermay be in the range of from 10-80 nm. An opaque layer need not block(i.e. reflect or absorb) 100 percent of the light from an excitationbeam. In some embodiments, an opaque layer may block at least 10 percentof incident light from an excitation beam; in other embodiments, anopaque layer may block at least 50 percent of incident light from anexcitation beam.

Opaque layers or coatings may be fabricated on solid state membranes bya variety of techniques known in the art. Material deposition techniquesmay be used including chemical vapor deposition, electrodeposition,epitaxy, thermal oxidation, physical vapor deposition, includingevaporation and sputtering, casting, and the like. In some embodiments,atomic layer deposition may be used, e.g. U.S. Pat. No. 6,464,842; Weiet al, Small, 6(13): 1406-1414 (2010), which are incorporated byreference.

In some embodiments, a 1-100 nm channel or aperture may be formedthrough a solid substrate, usually a planar substrate, such as amembrane, through which an analyte, such as single stranded DNA, isinduced to translocate. In other embodiments, a 2-50 nm channel oraperture is formed through a substrate; and in still other embodiments,a 2-30 nm, or a 2-20 nm, or a 3-30 nm, or a 3-20 nm, or a 3-10 nmchannel or aperture if formed through a substrate. The solid-stateapproach of generating nanopores offers robustness and durability aswell as the ability to tune the size and shape of the nanopore, theability to fabricate high-density arrays of nanopores on a wafer scale,superior mechanical, chemical and thermal characteristics compared withlipid-based systems, and the possibility of integrating with electronicor optical readout techniques. Biological nanopores on the other handprovide reproducible narrow bores, or lumens, especially in the 1-10nanometer range, as well as techniques for tailoring the physical and/orchemical properties of the nanopore and for directly or indirectlyattaching groups or elements, such as fluorescent labels, which may beFRET donors or acceptors, by conventional protein engineering methods.Protein nanopores typically rely on delicate lipid bilayers formechanical support, and the fabrication of solid-state nanopores withprecise dimensions remains challenging. In some embodiments, solid-statenanopores may be combined with a biological nanopore to form a so-called“hybrid” nanopore that overcomes some of these shortcomings, therebyproviding the precision of a biological pore protein with the stabilityof a solid state nanopore. For optical read out techniques a hybridnanopore provides a precise location of the nanopore which simplifiesthe data acquisition greatly.

In some embodiments, clusters may also be formed by disposing proteinnanopores in lipid bilayers supported by solid phase membrane containingan array of apertures. For example, such an array may comprise aperturesfabricated (e.g. drilled, etched, or the like) in solid phase support.The geometry of such apertures may vary depending on the fabricationtechniques employed. In some embodiments, each such aperture isassociated with, or encompassed by, a separate resolution limited area;however, in other embodiments, multiple apertures may be within the sameresolution limited area. The cross-sectional area of the apertures mayvary widely and may or may not be the same as between differentclusters, although such areas are usually substantially the same as aresult of conventional fabrication approaches. In some embodiments,apertures have a minimal linear dimension (e.g. diameter in the case ofcircular apertures) in the range of from 10 to 200 nm, or have areas inthe range of from about 100 to 3×10⁴ nm². Across the apertures may bedisposed a lipid bilayer. The distribution of protein nanopores peraperture may be varied, for example, by controlling the concentration ofprotein nanopores during inserting step. In such embodiments, clustersof nanopores may comprise a random number of nanopores. In someembodiments, in which protein nanopores insert randomly into apertures,clusters containing one or more apertures on average have a number ofprotein nanopores that is greater than zero; in other embodiments, suchclusters have a number of protein nanopores that is greater than 0.25;in other embodiments, such clusters have a number of protein nanoporesthat is greater than 0.5; in other embodiments, such clusters have anumber of protein nanopores that is greater than 0.75; in otherembodiments, such clusters have a number of protein nanopores that isgreater than 1.0.

In some embodiments, methods and devices of the invention comprise asolid phase membrane, such as a SiN membrane, having an array ofapertures therethrough providing communication between a first chamberand a second chamber (also sometimes referred to as a “cis chamber” anda “trans chamber”) and supporting a lipid bilayer on a surface facingthe second, or trans, chamber. In some embodiments, diameters of theaperture in such a solid phase membrane may be in the range of 10 to 200nm, or in the range of 20 to 100 nm. In some embodiments, such solidphase membranes further include protein nanopores inserted into thelipid bilayer in regions where such bilayer spans the apertures on thesurface facing the trans chamber. In some embodiments, such proteinnanopores are inserted from the cis side of the solid phase membraneusing techniques described herein. In some embodiments, such proteinnanopores have a structure identical to, or similar to, a-hemolysin inthat it comprises a barrel, or bore, along an axis and at one end has a“cap” structure and at the other end has a “stem” structure (using theterminology from Song et al, Science, 274: 1859-1866 (1996)). In someembodiments using such protein nanopores, insertion into the lipidbilayer results in the protein nanopore being oriented so that its capstructure is exposed to the cis chamber and its stem structure isexposed to the trans chamber.

In some embodiments, the present invention may employ hybrid nanoporesin clusters, particularly for optical-based nanopore sequencing ofpolynucleotides. Such nanopores comprise a solid-state orifice, oraperture, into which a protein biosensor, such as a protein nanopore, isstably inserted. A charged polymer may be attached to a protein nanopore(e.g. alpha hemolysin) by conventional protein engineering techniquesafter which an applied electric field may be used to guide a proteinnanopore into an aperture in a solid-state membrane. In someembodiments, the aperture in the solid-state substrate is selected to beslightly smaller than the protein, thereby preventing it fromtranslocating through the aperture. Instead, the protein will beembedded into the solid-state orifice.

In some embodiments, a donor fluorophore is attached to the proteinnanopore. This complex is then inserted into a solid-state aperture ornanohole (for example, 3-10 nm in diameter) by applying an electricfield across the solid state nanohole, or aperture, until the proteinnanopore is transported into the solid-state nanohole to form a hybridnanopore. The formation of the hybrid nanopore can be verified by (a)the inserted protein nanopore causing a drop in current based on apartial blockage of the solid-state nanohole and by (b) the opticaldetection of the donor fluorophore.

Solid state, or synthetic, nanopores may be preprared in a variety ofways, as exemplified in the references cited above. In some embodimentsa helium ion microscope may be used to drill the synthetic nanopores ina variety of materials, e.g. as disclosed by Yang et al, Nanotechnolgy,22: 285310 (2011), which is incorporated herein by reference. A chipthat supports one or more regions of a thin-film material, e.g. siliconnitride, that has been processed to be a free-standing membrane isintroduced to the helium ion microscope (HIM) chamber. HIM motorcontrols are used to bring a free-standing membrane into the path of theion beam while the microscope is set for low magnification. Beamparameters including focus and stigmation are adjusted at a regionadjacent to the free-standing membrane, but on the solid substrate. Oncethe parameters have been properly fixed, the chip position is moved suchthat the free-standing membrane region is centered on the ion beam scanregion and the beam is blanked. The HIM field of view is set to adimension (in pm) that is sufficient to contain the entire anticipatednanopore pattern and sufficient to be useful in future optical readout(i.e. dependent on optical magnification, camera resolution, etc.). Theion beam is then rastered once through the entire field of view at apixel dwell time that results in a total ion dose sufficient to removeall or most of the membrane autofluorescence. The field of view is thenset to the proper value (smaller than that used above) to performlithographically-defined milling of either a single nanopore or an arrayof nanopores. The pixel dwell time of the pattern is set to result innanopores of one or more predetermined diameters, determined through theuse of a calibration sample prior to sample processing. This entireprocess is repeated for each desired region on a single chip and/or foreach chip introduced into the HIM chamber.

In some embodiments, a nanopore may have one or more labels attached foruse in optically-based nanopore sequencing methods. The label may be amember of a Forster Resonance Energy Transfer (FRET) pair. Such labelsmay comprise organic fluorophores, chemiluminescent labels, quantumdots, metallic nanoparticles and/or fluorescent proteins. Target nucleicacids may have one distinct label per nucleotide. The labels attached tothe nucleotides may be selected from the group consisting of organicfluorophores. The label attachment site in the pore protein can begenerated by conventional protein engineering methods, e.g. a mutantprotein can be constructed that will allow the specific binding of thelabel. As an example, a cysteine residue may be inserted at the desiredposition of the protein which inserts a thiol (SH) group that can beused to attach a label. The cysteine can either replace a naturaloccurring amino acid or can be incorporated as an addition amino acid. Amaleimide-activated label is then covalently attached to the thiolresidue of the protein nanopore. In a preferred embodiment theattachment of the label to the protein nanopore or the label on thenucleic acid is reversible. By implementing a cleavable crosslinker, aneasily breakable chemical bond (e.g. an S-S bond or a pH labile bond) isintroduced and the label may be removed when the correspondingconditions are met.

Optically Based Nanopore Sequencing with FRET Signals

In some embodiments, a nanopore may be labeled with one or more quantumdots. In particular, in some embodiments, one or more quantum dots maybe attached to a nanopore, or attached to a solid phase support adjacentto (and within a FRET distance of an entrance or exit of a nanopore),and employed as donors in FRET reactions with acceptors on analytes.Such uses of quantum dots are well known and are described widely in thescientific and patent literature, such as, in U.S. Pat. Nos. 6,252,303;6,855,551; 7,235,361; and the like, which are incorporated herein byreference.

One example of a Quantum dot which may be utilized as a pore label is aCdTe quantum dot which can be synthesized in an aqueous solution. A CdTequantum dot may be functionalized with a nucleophilic group such asprimary amines, thiols or functional groups such as carboxylic acids. ACdTe quantum dot may include a mercaptopropionic acid capping ligand,which has a carboxylic acid functional group that may be utilized tocovalently link a quantum dot to a primary amine on the exterior of aprotein pore. The cross-linking reaction may be accomplished usingstandard cross-linking reagents (homo-bifunctional as well ashetero-bifunctional) which are known to those having ordinary skill inthe art of bioconjugation. Care may be taken to ensure that themodifications do not impair or substantially impair the translocation ofa nucleic acid through the nanopore. This may be achieved by varying thelength of the employed crosslinker molecule used to attach the donorlabel to the nanopore.

For example, the primary amine of the lysine residue 131 of the naturalalpha hemolysin protein (Song, L. et al., Science 274, (1996):1859-1866) may be used to covalently bind carboxy modified CdTe Quantumdots via 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride/N-hydroxysulfosuccinimide (EDC/NHS) coupling chemistry.Alternatively, amino acid 129 (threonine) may be exchanged intocysteine. Since there is no other cysteine residue in the natural alphahemolysin protein the thiol side group of the newly inserted cysteinemay be used to covalently attach other chemical moieties.

A biological polymer, e.g., a nucleic acid molecule or polymer, may belabeled with one or more acceptor labels. For a nucleic acid molecule,each of the four nucleotides or building blocks of a nucleic acidmolecule may be labeled with an acceptor label thereby creating alabeled (e.g., fluorescent) counterpart to each naturally occurringnucleotide. The acceptor label may be in the form of an energy acceptingmolecule which can be attached to one or more nucleotides on a portionor on the entire strand of a converted nucleic acid.

A variety of methods may be utilized to label the monomers ornucleotides of a nucleic acid molecule or polymer. A labeled nucleotidemay be incorporated into a nucleic acid during synthesis of a newnucleic acid using the original sample as a template (“labeling bysynthesis”). For example, the labeling of nucleic acid may be achievedvia PCR, whole genome amplification, rolling circle amplification,primer extension or the like or via various combinations and extensionsof the above methods known to persons having ordinary skill in the art.

A label may comprise a reactive group such as a nucleophile (amines,thiols etc.). Such nucleophiles, which are not present in naturalnucleic acids, can then be used to attach fluorescent labels via amineor thiol reactive chemistry such as NHS esters, maleimides, epoxy rings,isocyanates etc. Such nucleophile reactive fluorescent dyes (i.e.NHS-dyes) are readily commercially available from different sources. Anadvantage of labeling a nucleic acid with small nucleophiles lies in thehigh efficiency of incorporation of such labeled nucleotides when a“labeling by synthesis” approach is used. Bulky fluorescently labelednucleic acid building blocks may be poorly incorporated by polymerasesdue to steric hindrance of the labels during the polymerization processinto newly synthesized DNA.

Whenever two or more mutually quenching dyes are used, such dyes may beattached to DNA using orthogonal attachment chemistries. For example,NHS esters can be used to react very specifically with primary amines ormaleimides will react with thiol groups. Either primary amines (NH₂) orthiol (SH) modified nucleotides are commercially available. Theserelatively small modifications are readily incorporated in a polymerasemediated DNA synthesis and can be used for subsequent labeling reactionsusing either NHS or maleimide modified dyes. Guidance for selecting andusing such orthogonal linker chemistries may be found in Hermanson(cited above).

Additional orthogonal attachment chemistries for typical attachmentpositions include Huisgen-type cycloaddition for a copper-catalyzedreaction and an uncatalyzed reaction; alkene plus nitrile oxidecycloaddition, e.g. as disclosed in Gutsmiedl et al, Org. Lett., 11:2405-2408 (2009); Diels-Alder cycloaddition, e.g. disclosed in Seelig etal, Tetrahedron Lett., 38: 7729-7732 (1997); carbonyl ligation, e.g. asdisclosed in Casi et al, J. Am. Chem. Soc., 134: 5887-5892 (2012); Shaoet al J. Am. Chem. Soc., 117: 3893-3899 (1995); Rideout, Science, 233:561-563 (1986); Michael addition, e.g. disclosed in Brinkley,Bioconjugate Chemistry, 3: 2-13 (1992); native chemical ligation, e.g.disclosed in Schuler et al, Bioconjugate Chemistry, 13: 1039-1043(2002); Dawson et al, Science, 266: 776-779 (1994); or amide formationvia an active ester, e.g. disclosed in Hermanson (cited above).

A combination of 1, 2, 3 or 4 nucleotides in a nucleic acid strand maybe exchanged with their labeled counterpart. The various combinations oflabeled nucleotides can be sequenced in parallel, e.g., labeling asource nucleic acid or DNA with combinations of 2 labeled nucleotides inaddition to the four single labeled samples, which will result in atotal of 10 differently labeled sample nucleic acid molecules or DNAs(G, A, T, C, GA, GT, GC, AT, AC, TC). The resulting sequence pattern mayallow for a more accurate sequence alignment due to overlappingnucleotide positions in the redundant sequence read-out. In someembodiments, a polymer, such as a polynucleotide or polypeptide, may belabeled with a single fluorescent label attached to a single kind ofmonomer, for example, every T (or substantially every T) of apolynucleotide is labeled with a fluorescent label, e.g. a cyanine dye.In such embodiments, a collection, or sequence, of fluorescent signalsfrom the polymer may form a signature or fingerprint for the particularpolymer. In some such embodiments, such fingerprints may or may notprovide enough information for a sequence of monomers to be determined.

In some embodiments, a feature of the invention is the labeling ofsubstantially all monomers of a polymer analyte with fluorescent dyes orlabels that are members of a mutually quenching set. The use of the term“substantially all” in reference to labeling polymer analytes is toacknowledge that chemical and enzymatic labeling techniques aretypically less than 100 percent efficient. In some embodiments,“substantially all” means at least 80 percent of all monomer havefluorescent labels attached. In other embodiments, “substantially all”means at least 90 percent of all monomer have fluorescent labelsattached. In other embodiments, “substantially all” means at least 95percent of all monomer have fluorescent labels attached.

A method for sequencing a polymer, such as a nucleic acid moleculeincludes providing a nanopore or pore protein (or a synthetic pore)inserted in a membrane or membrane like structure or other substrate.The base or other portion of the pore may be modified with one or morepore labels. The base may refer to the Trans side of the pore.Optionally, the Cis and/or Trans side of the pore may be modified withone or more pore labels. Nucleic acid polymers to be analyzed orsequenced may be used as a template for producing a labeled version ofthe nucleic acid polymer, in which one of the four nucleotides or up toall four nucleotides in the resulting polymer is/are replaced with thenucleotide's labeled analogue(s). An electric field is applied to thenanopore which forces the labeled nucleic acid polymer through thenanopore, while an external monochromatic or other light source may beused to illuminate the nanopore, thereby exciting the pore label. As,after or before labeled nucleotides of the nucleic acid pass through,exit or enter the nanopore, energy is transferred from the pore label toa nucleotide label, which results in emission of lower energy radiation.The nucleotide label radiation is then detected by a confocal microscopesetup or other optical detection system or light microscopy systemcapable of single molecule detection known to people having ordinaryskill in the art. Examples of such detection systems include but are notlimited to confocal microscopy, epi-illumination fluorescencemicroscopy, and the like. In some embodiments, epi-illuminationfluorescence microscopy is employed.

Energy may be transferred from a pore or nanopore donor label (e.g., aQuantum Dot) to an acceptor label on a polymer (e.g., a nucleic acid)when an acceptor label of an acceptor labeled monomer (e.g., nucleotide)of the polymer interacts with the donor label as, after or before thelabeled monomer exits, enters or passes through a nanopore. For example,the donor label may be positioned on or attached to the nanopore on thecis or trans side or surface of the nanopore such that the interactionor energy transfer between the donor label and acceptor label does nottake place until the labeled monomer exits the nanopore and comes intothe vicinity or proximity of the donor label outside of the nanoporechannel or opening. As a result, interaction between the labels, energytransfer from the donor label to the acceptor label, emission of energyfrom the acceptor label and/or measurement or detection of an emissionof energy from the acceptor label may take place outside of the passage,channel or opening running through the nanopore, e.g., within a cis ortrans chamber on the cis or trans sides of a nanopore. The measurementor detection of the energy emitted from the acceptor label of a monomermay be utilized to identify the monomer.

The nanopore label may be positioned outside of the passage, channel oropening of the nanopore such that the label may be visible or exposed tofacilitate excitation or illumination of the label. The interaction andenergy transfer between a donor label and accepter label and theemission of energy from the acceptor label as a result of the energytransfer may take place outside of the passage, channel or opening ofthe nanopore. This may facilitate ease and accuracy of the detection ormeasurement of energy or light emission from the acceptor label, e.g.,via an optical detection or measurement device.

A donor label may be attached in various manners and/or at various siteson a nanopore. For example, a donor label may be directly or indirectlyattached or connected to a portion or unit of the nanopore.Alternatively, a donor label may be positioned adjacent to a nanopore.

Each acceptor labeled monomer (e.g., nucleotide) of a polymer (e.g.,nucleic acid) can interact sequentially with a donor label positioned onor next to or attached directly or indirectly to the exit of a nanoporeor channel through which the polymer is translocated. The interactionbetween the donor and acceptor labels may take place outside of thenanopore channel or opening, e.g., after the acceptor labeled monomerexits the nanopore or before the monomer enters the nanopore. Theinteraction may take place within or partially within the nanoporechannel or opening, e.g., while the acceptor labeled monomer passesthrough, enters or exits the nanopore.

When one of the four nucleotides of a nucleic acid is labeled, the timedependent signal arising from the single nucleotide label emission isconverted into a sequence corresponding to the positions of the labelednucleotide in the nucleic acid sequence. The process is then repeatedfor each of the four nucleotides in separate samples and the fourpartial sequences are then aligned to assemble an entire nucleic acidsequence.

When multi-color labeled nucleic acid (DNA) sequences are analyzed, theenergy transfer from one or more donor labels to each of the fourdistinct acceptor labels that may exist on a nucleic acid molecule mayresult in light emission at four distinct wavelengths or colors (eachassociated with one of the four nucleotides) which allows for a directsequence read-out.

A donor label (also sometimes referred to herein as a “pore label”) maybe placed as close as possible to the aperture (for example, at theexit) of a nanopore without causing an occlusion that impairstranslocation of a nucleic acid through the nanopore. A pore label mayhave a variety of suitable properties and/or characteristics. Forexample, a pore label may have energy absorption properties meetingparticular requirements. A pore label may have a large radiation energyabsorption cross-section, ranging, for example, from about 0 to 1000 nmor from about 200 to 500 nm. A pore label may absorb radiation within aspecific energy range that is higher than the energy absorption of thenucleic acid label, such as an acceptor label. The absorption energy ofthe pore label may be tuned with respect to the absorption energy of anucleic acid label in order to control the distance at which energytransfer may occur between the two labels. A pore label may be stableand functional for at least 106 to 109 excitation and energy transfercycles.

In some embodiments, a device for analyzing polymers each having opticallabels attached to a sequence of monomers may comprise the followingelements: (a) a nanopore array in a solid phase membrane separating afirst chamber and a second chamber, wherein nanopores of the nanoporearray each provide fluid communication between the first chamber and thesecond chamber and are arranged in clusters such that each differentcluster of nanopores is disposed within a different resolution limitedarea and such that each cluster comprises a number of nanopores that iseither greater than one or is a random variable with an average valuegreater than zero; (b) a polymer translocating system for movingpolymers in the first chamber to the second chamber through thenanopores of the nanopore array; and (c) a detection system forcollecting optical signals generated by optical labels attached topolymers whenever an optical label exits a nanopore within a resolutionlimited area.

Optically Based Nanopore Sequencing with Self-Quenching Dyes And/OrQuenching Agents

In one aspect, the invention includes the using nanopore arrays withopaque layers and direct illumination of fluorescently labeledpolynucleotides for sequence determination. In some embodiments, suchapplications includes the use of fluorescent quenching, and fluorescentsignaling to sequentially identify nucleotides of fluorescently labeledpolynucleotide analytes. Such analysis of polynucleotide analytes may becarried out on pluralities of polynucleotides in parallel at the sametime, for example, by using an array of nanopores containing an opaquelayer. In some embodiments, nucleotides are labeled with fluorescentlabels that are capable of at least three states while attached to apolynucleotide: (i) A substantially quenched state wherein fluorescenceof an attached fluorescent label is quenched by a fluorescent label onan immediately adjacent monomer or by interaction with a quenchingagent; for example, a fluorescent label attached to a polynucleotide inaccordance with the invention is substantially quenched when the labeledpolynucleotide is free in conventional aqueous solutions or buffers forstudying and manipulating the polynucleotide. (ii) A stericallyconstrained state while a labeled polynucleotide is translocatingthrough a nanopore such that the free-solution movements or alignmentsof attached fluorescent labels are disrupted or limited so that there islittle or no detectable fluorescent signal generated from thefluorescent label. (iii) A transition state wherein fluorescent labelsattached to a polynucleotide transition from the sterically constrainedstate to a quenched state as the nucleotide of the fluorescent labelexits the nanopore (during a “transition interval” or “interval”).During the transition interval a fluorescent label (on an otherwisesubstantially fully labeled and self-quenched or quenchedpolynucleotide) is capable of generating a detectable fluorescent signaland that the number of exiting labels contributing to a measured signalmay be (at least in part) controlled by controlling the translocationspeed of the labeled polynucleotide. If translocation speed (e.g.nucleotides exiting a nanopore per msec) is higher than the transitionrate (from signal-capable to quenched, i.e. the quenching rate), thenmeasured fluorescent signals, or signal samples, may containcontributions from more than one label.

Without the intention of being limited by any theory underlying theabove process, it is believed that the fluorescent signal generatedduring the transition interval is due to the presence of one or morefreely rotatable dipoles of the fluorescent labels that emerged from ananopore, which renders the fluorescent labels capable of generating afluorescent signal, for example, after direct excitation or viaexcitation via FRET. In some embodiments, the polynucleotide is a singlestranded polynucleotide, such as, DNA or RNA, but especially a singlestranded DNA. In some embodiments, the invention includes a method fordetermining a nucleotide sequence of a polynucleotide by recordingsignals generated by fluorescent labels as they exit a nanopore one at atime as a polynucleotide translocates through the nanopore. Atranslocation speed may be selected to maximize the likelihood thatmeasured fluorescent signals comprise fluorescence from substantiallyonly a single label, wherein such selection may be made either byreal-time adjustment of parameters controllable during operation (suchas the voltage across the nanopores, temperature, or the like) or bypredetermined instrument set-up (e.g. reaction buffer viscosity, ionconcentration, or the like). Upon exit, each attached fluorescent labeltransitions during a transition interval from a constrained state in thenanopore to a quenched state on the polynucleotide in free solution.During the transition interval the label is capable of generating afluorescent signal which can be measured. In other words, in someembodiments, a step of the method may comprise exciting each fluorescentlabel as it is transitioning from a constrained state in the nanopore toa quenched state on the polymer in free solution. As mentioned above,during this transition interval or period a fluorescent label is capableof emitting a detectable fluorescent signal indicative of the nucleotideto which it is attached.

In some embodiments, “substantially quenched” as used above means afluorescent label generates a fluorescent signal at least thirty percentreduced from a signal generated under the same conditions, but withoutadjacent mutually quenching labels. In some embodiments, “substantiallyquenched” as used above means a fluorescent label generates afluorescent signal at least fifty percent reduced from a signalgenerated under the same conditions, but without adjacent mutuallyquenching labels.

The above concepts are illustrated in FIGS. 4A-4B, whichdiagrammatically show labeled polynucleotide (4000) translocatingthrough nanopore (4002). Labeled polynucleotide (4000) comprises twolabels “a” and “b” (for example, which may correspond to dC beinglabeled with “a” and dA, dG and dT being labeled with “b”, or the like).Labels of nucleotides free of nanopore (4002) are quenched, either byinteraction with other labels (4011) or by action of quenching agents(not shown). Labels of nucleotides inside of nanopore (4002) areconstrained and/or oriented (4014) so that they produce no detectablesignal during all or part of their transit through the nanopore. Asnucleotides of labeled polynucleotide (4000) emerge from exit (4015) ofnanopore (4002) they become capable of being excited by excitation beam(4010) and generating a detectable signal for an interval prior to beingquenched. If translocation speed V₁ is high then the distance (4008)traveled by a nucleotide prior to quenching may exceed theinter-nucleotide distance of polynucleotide (4000) so that more than onelabel (shown in FIG. 4A) contributes fluorescence to a fluorescentsignal collected by detector (4018), i.e. a measured fluorescent signal.If translocation speed V₂ is low then the distance (4008) traveled by anucleotide prior to quenching may approximately equal or be less thanthe inter-nucleotide distance of polynucleotide (4000) so that no morethan one label (shown in FIG. 4B) contributes fluorescence to afluorescent signal collected by detector (4018), i.e. a measuredfluorescent signal. Since the distance between adjacent labels is belowthe diffraction limit of excitation light (4010) no information isobtained about the ordering of the labels, although there are approachesto deduce such information using specialized algorithms, e.g. Andersonet al, U.S. provisional patent application 62/322343; Timp et al,Biophys. J., 102: L37-L39 (2012); Carson et al, Nanotechnology, 26:074004 (2015). In the case of optical detection using fluorescent labelswith distinct emission bands, measured fluorescent signals may beseparated into two or more channels, e.g. using bandpass filters, inorder to assess the relative contributions of fluorescence from multiplelabels. However, as the number of fluorescent labels contributingfluorescence increases, e.g. 3, 4, or more, the difficulty indetermining a correct ordering of nucleotides increases. The signalintensities for two channels, e.g. corresponding to emission maxima oftwo fluorescent labels, is illustrated in FIG. 4A (4031 and 4032) wheretwo fluorescent labels contribute to a measured signal and in FIG. 4B(4041 and 4042) where a single fluorescent label contributes to ameasured signal. Intensity values represented by solid lines, e.g. 4033,are from label “a,” and intensity values represented by dashed lines,e.g. 4036, are from label “b”. The presence of solid and dashed lines inboth channels of FIG. 4A reflects overlapping emission bands of thefluorescent labels, which when collected together complicates analysisbecause amounts of a measured intensity are from both labels. In FIG.4B, where only a single fluorescent label contributes to the measuredsignal, intensity values do not contain contributions due to overlappingemission bands of other labels, thereby making label (and thereforenucleotide) determination easier.

The role of translocation speed of polynucleotides through nanopores andthe need for its control have been appreciated in the field of nanoporetechnology wherein changes in electric current are use to identifytranslocating analytes. A wide variety of methods have been used tocontrol translocation speed, which include both methods that can beadjusted in real-time without significant difficulty (e.g. voltagepotential across nanopores, temperature, and the like) and methods thatcan be adjusted during operation only with difficulty (reaction bufferviscosity, presence or absence of charged side chains in the bore of aprotein nanopore, ionic composition and concentration of the reactionbuffer, velocity-retarding groups attached or hybridized topolynucleotide analytes, molecular motors, and the like), e.g. Bates etal, Biophysical J., 84: 2366-2372 (2003); Carson et al, Nanotechnology,26(7): 074004 (2015); Yeh et al, Electrophoresis, 33(23): 58-65 (2012);Meller, J. Phys. Cond. Matter, 15: R581-R607 (2003); Luan et al,Nanoscale, 4(4): 1068-1077 (2012); Keyser, J. R. Soc. Interface, 8:1369-1378 (2011); and the like, which are incorporated herein byreference. In some embodiments, a step or steps may be included foractive control of translocation speed while a method of the invention isbeing implemented, e.g. voltage potential, temperature, or the like; inother embodiments, a step or steps are included that determine atranslocation speed that is not actively controlled or changed while amethod of the invention is being implemented, e.g. reaction bufferviscosity, ionic concentration, and the like. In regard to the latter,in some embodiments, a translocation speed is selected by providing areaction buffer having a concentration of glycerol, or equivalentreagent, in the range of from 1 to 60 percent.

In regard to the former embodiments (with real-time translocation speedadjustment), a measure of whether one or more than one label iscontributing fluorescence to measured signals may be based on thedistribution of fluorescence intensity among a plurality of channelsover which fluorescence is collected. Typically the plurality ofchannels include 2, 3, or 4 channels corresponding to the emission bandsof the fluorescent labels used. In a measured sample of fluorescenceemanating from a region adjacent to a nanopore exit, if only a singlelabel contributes to a measured signal, the relative distribution ofsignal intensity among the different channels (e.g. 4 channels) could berepresented ideally as (1,0,0,0); (0,1,0,0); (0,0,1,0) or (0,0,0,1). Onthe other hand, if more than one label contributed to a measuredfluorescent signal, the relative distributions would include non-zerovalues in more than one channel, with a worse case being four differentlabels contributing equally, which would appear as (0.25,0.25,0.25,0.25)in the above representation. A measure which would vary monotonicallybetween a maximum value corresponding to relative intensitydistributions (1,0,0,0); (0,1,0,0); (0,0,1,0) or (0,0,0,1) and a minimumvalue corresponding to a relative intensity distribution of(0.25,0.25,0.25,0.25) may be used for controlling in real-time atranslocation speed. For example, an initial translocation speed couldbe lowered based on the value of such a measure that was near itsminimum. Such lowering may be implemented, for example, by lowering apotential voltage across the nanopores by a predetermined amount, afterwhich the measure could be re-calculated. Such steps could be repeateduntil the process was optimized.

As mentioned above, translocation speeds depend in part on the voltagedifference (or electrical field strength) across a nanopore andconditions in the reaction mixture, or buffer, of a first chamber wherepolynucleotides are exposed to the nanopores (e.g. disposed in a solidphase membrane making up one wall of the first chamber). Polynucleotidecapture rates by nanopores depend on concentration of suchpolynucleotides. In some embodiments, conventional reaction mixtureconditions for nanopore sequencing may be employed with the invention(for controlling translocatin speed by varying voltage potential acrossnanopores), for example, 1M KCl (or equivalent salt, such as NaCl, LiCl,or the like) and a pH buffering system (which, for example, ensures thatproteins being used, e.g. protein nanopores, nucleases, or the like, arenot denatured). In some embodiments, a pH buffering system may be usedto keep the pH substantially constant at a value in the range of 6.8 to8.8. In some embodiments, a voltage difference across the nanopores maybe in the range of from 70 to 200 mV. In other embodiments, a voltagedifference across the nanopores may be in the range of from 80 to 150mV. An appropriate voltage for operation may be selected usingconventional measurement techniques. Current (or voltage) across ananopore may readily be measured using commercially availableinstruments. A voltage difference may be selected so that translocationspeed is within a desired range. In some embodiments, a range oftranslocation speeds comprises those speeds less than 1000 nucleotidesper second. In other embodiments, a range of translocation speeds isfrom 10 to 800 nucleotides per second; in other embodiments, a range oftranslocation speeds is from 10 to 600 nucleotides per second; in otherembodiments, a range of translocation speeds is from 200 to 800nucleotides per second; in other embodiments, a range of translocationspeeds is from 200 to 500 nucleotides per second. Likewise, otherfactors affecting translocation speed, e.g. temperature, viscosity, ionconcentration, charged side chains in the bore of a protein nanopore,and the like, may be selected to obtain translocation speeds in theranges cited above.

In some embodiments, a device for implementing the above methods forsingle stranded nucleic acids typically includes providing a set ofelectrodes for establishing an electric field across the nanopores(which may comprise an array). Single stranded nucleic acids are exposedto nanopores by placing them in an electrolyte (i.e. reaction buffer) ina first chamber, which is configured as the “cis” side of the layeredmembrane by placement of a negative electrode in the chamber. Uponapplication of an electric field, the negatively single stranded nucleicacids are captured by nanopores and translocated to a second chamber onthe other side of the layered membrane, which is configured as the“trans” side of membrane by placement of a positive electrode in thechamber. As mentioned above, the speed of translocation depends in parton the ionic strength of the electrolytes in the first and secondchambers and the applied voltage across the nanopores. In opticallybased detection, a translocation speed may be selected by preliminarycalibration measurements, for example, using predetermined standards oflabeled single stranded nucleic acids that generate signals at differentexpected rates per nanopore for different voltages. Thus, for DNAsequencing applications, an initial translocation speed may be selectedbased on the signal rates from such calibration measurements, as well asthe measure based on relative signal intensity distribution discussedabove. Consequently, from such measurements a voltage may be selectedthat permits, or maximizes, reliable nucleotide identifications, forexample, over an array of nanopores. In some embodiments, suchcalibrations may be made using nucleic acids from the sample oftemplates being analyzed (instead of, or in addition to, predeterminedstandard sequences). In some embodiments, such calibrations may becarried out in real time during a sequencing run and the applied voltagemay be modified in real time based on such measurements, for example, tomaximize the acquisition of nucleotide-specific signals.

Embodiments Employing Mutually and Self-Quenching Labels

As mentioned above, in some embodiments, self- and mutually quenchingfluorescent labels may be used in addition to quenching agents in orderto reduce fluorescent emissions outside of those from labels onnucleotides exiting nanopores. Use of such fluorescent labels isdisclosed in U.S. patent publication 2016/0122812, which is incorporatedby reference. In some embodiments, monomers are labeled with fluorescentlabels that are capable of at least three states while attached to atarget polynucleotide: (i) A substantially quenched state whereinfluorescence of an attached fluorescent label is quenched by afluorescent label on an immediately adjacent monomer; for example, afluorescent label attached to a polynucleotide in accordance with theinvention is substantially quenched when the labeled polynucleotide isfree in conventional aqueous solution for studying and manipulating thepolynucleotide. (ii) A sterically constrained state wherein a labeledpolynucleotide is translocating through a nanopore such that thefree-solution movements or alignments of an attached fluorescent labelis disrupted or limited so that there is little or no detectablefluorescent signal generated from the fluorescent label. (iii) Atransition state wherein a fluorescent label attached to apolynucleotide transitions from the sterically constrained state to thequenched state as the fluorescent label exits the nanopore (during a“transition interval”) while the polynucleotide translocates through thenanopore.

In part, this example is an application of the discovery that during thetransition interval a fluorescent label (on an otherwise substantiallyfully labeled and self-quenched polynucleotide) is capable of generatinga detectable fluorescent signal. Without the intention of being limitedby any theory underlying this discovery, it is believed that thefluorescent signal generated during the transition interval is due tothe presence of a freely rotatable dipole in the fluorescent labelemerging from the nanopore, which renders the fluorescent labeltemporarily capable of generating a fluorescent signal, for example,after direct excitation or via FRET. In both the sterically constrainedstate as well as the quenched state, the dipoles are limited in theirrotational freedom thereby reducing or limiting the number of emittedphotons. In some embodiments, the polynucleotide is a polynucleotide,usually a single stranded polynucleotide, such as, DNA or RNA, butespecially single stranded DNA. In some embodiments, the inventionincludes a method for determining a nucleotide sequence of apolynucleotide by recording signals generated by attached fluorescentlabels as they exit a nanopore one at a time as a polynucleotidetranslocates through the nanopore. Upon exit, each attached fluorescentlabel transitions during a transition interval from a constrained statein the nanopore to a quenched state on the polynucleotide in freesolution. In other words, in some embodiments, a step of the method ofthe invention comprises exciting each fluorescent label as it istransitioning from a constrained state in the nanopore to a quenchedstate on the polynucleotide in free solution. As mentioned above, duringthis transition interval or period the fluorescent label is capable ofemitting a detectable fluorescent signal indicative of the nucleotide itis attached to.

In some embodiments, the invention includes an application of thediscovery that fluorescent labels and nanopores may be selected so thatduring translocation of a polynucleotide through a nanopore fluorescentlabels attached to monomers are forced into a constrained state in whichthey are incapable (or substantially incapable) of producing adetectable fluorescent signal. In some embodiments, nanopores areselected that have a bore, or lumen, with a diameter in the range offrom 1 to 4 nm; in other embodiments, nanopores are selected that have abore or lumen with a diameter in the range of from 2 to 3 nm. In someembodiments, such bore diameters are provided by a protein nanopore. Insome embodiments, such nanopores are used to force fluorescent labelsinto a constrained state in accordance with the invention, so thatwhenever a fluorescent label exits a nanopore, it transitions from beingsubstantially incapable of generating a fluorescent signal to beingdetectable and identifiable by a fluorescent signal it can be induced toemit. Thus, fluorescent labels attached to each of a sequence ofmonomers of a polynucleotide may be detected in sequence as theysuddenly generate a fluorescent signal in a region immediately adjacentto a nanopore exit (a “transition zone” or “transition volume” or“detection zone”). In some embodiments, organic fluorescent dyes areused as fluorescent labels with nanopores of the above diameters. Insome embodiments, at least one such organic fluorescent dye is selectedfrom the set consisting of xanthene dyes, rhodamine dyes and cyaninedyes. Some embodiments for determining a monomer sequence of apolynucleotide may be carried out with the following steps: (a)translocating a polynucleotide through a nanopore, wherein monomers ofthe polynucleotide are labeled with fluorescent labels wherein thenanopore constrains fluorescent labels within its bore into aconstrained state such that substantially no detectable fluorescentsignal is generated therein; (b) exciting the fluorescent label of eachmonomer upon exiting the nanopore; (c) measuring a fluorescent signal ina detection zone generated by the exiting fluorescent label to identifythe monomer to which the fluorescent label is attached; (d) quenchingfluorescent signals from excited fluorescent labels outside of thedetection zone, and (d) determining a monomer sequence of thepolynucleotide from a sequence of fluorescent signals. In furtherembodiments, fluorescent labels are acceptors of a FRET pair and one ormore donors of the FRET pair are attached to the nanopore within a FRETdistance of the exit.

In some embodiments, “substantially quenched” as used above means afluorescent label generates a fluorescent signal at least thirty percentreduced from a signal generated under the same conditions, but withoutadjacent mutually quenching labels. In some embodiments, “substantiallyquenched” as used above means a fluorescent label generates afluorescent signal at least fifty percent reduced from a signalgenerated under the same conditions, but without adjacent mutuallyquenching labels.

In some embodiments, a nucleotide sequence of a target polynucleotide isdetermined by carrying out four separate reactions in which copies ofthe target polynucleotide have each of its four different kinds ofnucleotide (A, C, G and T) labeled with a single fluorescent label. In avariant of such embodiments, a nucleotide sequence of a targetpolynucleotide is determined by carrying out four separate reactions inwhich copies of the target polynucleotide have each of its fourdifferent kinds of nucleotide (A, C, G and T) labeled with onefluorescent label while at the same time the other nucleotides on thesame target polynucleotide are labeled with a second fluorescent label.For example, if a first fluorescent label is attached to A's of thetarget polynucleotide in a first reaction, then a second fluorescentlabel is attached to C's, G's and T's (i.e. to the “not-A” nucleotides)of the target polynucleotides in the first reaction. Likewise, incontinuance of the example, in a second reaction, the first label isattached to C's of the target polynucleotide and the second fluorescentlabel is attached to A's, G's and T's (i.e. to the “not-C” nucleotides)of the target polynucleotide. And so on, for nucleotides G and T.

The same labeling scheme may be expressed in terms of conventionalterminology for subsets of nucleotide types; thus, in the above example,in a first reaction, a first fluorescent label is attached to A's and asecond fluorescent label is attached to B's; in a second reaction, afirst fluorescent label is attached to C's and a second fluorescentlabel is attached to D's; in a third reaction, a first fluorescent labelis attached to G's and a second fluorescent label is attached to H's;and in a fourth reaction, a first fluorescent label is attached to T'sand a second fluorescent label is attached to V's.

In some embodiments, a polymer, such as a polynucleotide or peptide, maybe labeled with a single fluorescent label attached to a single kind ofmonomer, for example, every T (or substantially every T) of apolynucleotide is labeled with a fluorescent label, e.g. a cyanine dye.In such embodiments, a collection, or sequence, of fluorescent signalsfrom the polynucleotide may form a signature or fingerprint for theparticular polynucleotide. In some such embodiments, such fingerprintsmay or may not provide enough information for a sequence of monomers tobe determined.

In some embodiments, a feature of the invention is the labeling ofsubstantially all monomers of a polynucleotide analyte with fluorescentdyes or labels that are members of a mutually quenching set. The use ofthe term “substantially all” in reference to labeling polynucleotideanalytes is to acknowledge that chemical and enzymatic labelingtechniques are typically less than 100 percent efficient. In someembodiments, “substantially all” means at least 80 percent of allmonomer have fluorescent labels attached. In other embodiments,“substantially all” means at least 90 percent of all monomer havefluorescent labels attached. In other embodiments, “substantially all”means at least 95 percent of all monomer have fluorescent labelsattached. Mutually quenching sets of fluorescent dyes have the followingproperties: (i) each member quenches fluorescence of every member (forexample, by FRET or by static or contact mechanisms), and (ii) eachmember generates a distinct fluorescent signal when excited and when ina non-quenched state. That is, if a mutually quenching set consists oftwo dyes, D1 and D2, then (i) D1 is self-quenched (e.g. by contactquenching with another D1 molecule) and it is quenched by D2 (e.g. bycontact quenching) and (ii) D2 is self-quenched (e.g. by contactquenching with another D2 molecule) and it is quenched by D1 (e.g. bycontact quenching). Guidance for selecting fluorescent dyes or labelsfor mutually quenching sets may be found in the following references,which are incorporated herein by reference: Johansson, Methods inMolecular Biology, 335: 17-29 (2006); Marras et al, Nucleic AcidsResearch, 30: e122 (2002); and the like. In some embodiments, members ofa mutually quenching set comprise organic fluorescent dyes thatcomponents or moieties capable of stacking interactions, such asaromatic ring structures. Exemplary mutually quenching sets offluorescent dyes, or labels, may be selected from rhodamine dyes,fluorescein dyes and cyanine dyes. In one embodiment, a mutuallyquenching set may comprise the rhodamine dye, TAMRA, and the fluoresceindye, FAM. In another embodiment, mutually quenching sets of fluorescentdyes may be formed by selecting two or more dyes from the groupconsisting of Oregon Green 488, Fluorescein-EX, fluoresceinisothiocyanate, Rhodamine Red-X, Lissamine rhodamine B, Calcein,Fluorescein, Rhodamine, one or more BODIPY dyes, Texas Red, Oregon Green514, and one or more Alexa Fluors. Respresentative BODIPY dyes includeBODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY630/650 and BODIPY 650/665. Representative Alexa Fluors include AlexaFluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,635, 647, 660, 680, 700, 750 and 790.

As above, in some embodiments, a monomer sequence of a targetpolynucleotide is determined by carrying out separate reactions (one foreach kind of monomer) in which copies of the target polynucleotide haveeach different kind of monomer labeled with a mutually- orself-quenching fluorescent label. In other embodiments, a monomersequence of a target polynucleotide is determined by carrying outseparate reactions (one for each kind of monomer) in which copies of thetarget polynucleotide have each different kind of monomer labeled with adifferent mutually quenching fluorescent label selected from the samemutually quenching set. In embodiments in which a mutually quenching setcontains only two dyes, then a selected monomer (say, monomer X) islabeled with a first mutually quenching dye and every other kind ofmonomer (i.e., not-monomer X) is labeled with a second mutuallyquenching dye from the same set. Thus, steps of the embodiment generatea sequence of two different fluorescent signals, one indicating monomerX and another indicating not-monomer X.

In some embodiments, a single fluorescent label (for example, attachedto a single kind of monomer in a polynucleotide comprising multiplekinds of monomers) may be used that is self-quenching when attached toadjacent monomers (of the same kind) on a polynucleotide, such asadjacent nucleotides of a polynucleotide. Exemplary self-quenchingfluorescent labels include, but are not limited to, Oregon Green 488,fluorescein-EX, FITC, Rhodamine Red-X, Lissamine rhodamine B, calcein,fluorescein, rhodamine, BODIPYS, and Texas Red, e.g. which are disclosedin Molecular Probes Handbook, 11th Edition (2010).

Embodiments Employing Quenching Agents

As explained more fully below, a large variety of non-fluorescentquenching agents are available for use with the invention, which includederivatives of many well-known organic dyes, such as asymmetric cyaninedyes, as well as conjugates of such compounds and oligonucleotidesand/or analogs thereof. Quenching agents may be disposed in either thecis chamber, the trans chamber, or both. FIG. 4C illustrates anembodiment which includes the following elements: protein nanopore (400)disposed in lipid bilayer (402); epi-illumination of fluorescent labelswith opaque layer (408) in solid phase membrane (406) to prevent orreduce background fluorescence; and quenching agents (410) disposed intrans chamber (426). As above, polynucleotide (420) with fluorescentlylabeled nucleotides (labels being indicated by “f”, as with (422)) istranslocated through nanopore (400) from cis chamber (424) to transchamber (426). Oligonucleotide quenchers (410) are disposed in transchamber (426) under conditions (e.g. concentration, temperature, saltconcentration, and the like) that permits hybridization ofoligonucleotide quenchers (428) to portions of polynucleotide (420)emerging from nanopore (400). Nanopore (400) may be selected so thatsignals from fluorescent labels are suppressed during transit of thenanopore as described in Huber et al, U.S. patent publication US2016/0076091, which is incorporated herein by reference. Thus, whenlabeled nucleotides emerge from nanopore (400) in region (428) theybecome unsuppressed and capable of generating a signal. With most if notall forms of direct illumination (e.g. non-FRET) such emerged labelswould continue to emit fluorescence as they travel further into transchamber (426), thereby contributing greatly to a collected signal. Withquenching agents in trans chamber (426) that bind to the emergingpolynucleotide, such emissions can be significantly reduced and candefine detection zone (428) from which collected signals can be analyzedto give nucleotide sequence information about polynucleotide (420). Insome embodiments, a fluorescent signal from a single fluorescent labelis detected from detection zone (428) during a detection period as thelabeled polynucleotide moves through the detection zone. In otherembodiments, a plurality of fluorescent signals is collected from aplurality of fluorescent labels in detection zone (428) during apredetermined time period. In some embodiments, such detection period isless than 1 msec, or less than 0.1 msec, or less than 0.01 msec. In someembodiments, such detection perior is at least 0.01 msec, or at least0.1 msec, or at least 0.5 msec.

Quenching agents of the invention comprise any compound (or set ofcompounds) that under nanopore sequencing conditions is (i)substantially non-fluorescent, (ii) binds to single stranded nucleicacids, particularly single stranded DNA, and (iii) absorbs excitationenergy from other molecules non-radiatively and releases itnon-radiatively. In some embodiments, quenching agents further bindnon-covalently to single stranded DNA. A large variety of quenchingcompounds are available for use with the invention including, but notlimited to, non-fluorescent derivatives of common synthetic dyes such ascyanine and xanthene dyes, as described more fully below. Guidance inselecting quenching compounds may be found in U.S. Pat. Nos. 6,323,337;6,750,024 and like references, which are incorporated herein byreference.

In some embodiments, a quenching agent may be a single stranded DNAbinding dye that has been covalently modified with a heavy atom that isknown to quench fluorescence (such as bromine or iodine), or covalentlymodified with other groups known to quench fluorescence, such as a nitrogroup or a azo group. An example of dye that is known to bind singlestranded DNA is Sybr Green (Zipper et al, (2004), Nucleic AcidsResearch. 32 (12)). Incorporation of a nitro, bromine, iodine, and/orazo groups into the cynanine Sybr Green structure provides a singlestranded DNA binding group moiety that will quench fluorescent labelsthat might be present on a DNA.

In some embodiments, quenching agents comprise a binding moiety and oneor more quenching moieties. Binding moieties may include any compoundthat binds to single stranded nucleic acids without substantial sequencespecificity. Binding moieties may comprise peptides or oligonucleotidesor analogs of either having modified linkages and/or monomers.Oligonucleotides and their analogs may provide binding topolynucleotides via duplex formation or via non-base paired aptamericbinding. In some embodiments, binding moieties comprise anoligonucleotide or analog thereof having a length in the range of from 6to 60 nucleotides. Such oligonucleotides or analogs may be conjugated toone quenching moiety or to a plurality of quenching moieties. In someembodiments, the plurality of quenching moieties conjugated to eacholigonucleotide or analog is 2 or 3. Quenching moieties conjugated to abinding moiety may be the same or different. In some embodiments,whenever a binding moiety is an oligonucleotide or analog, two quenchingmoieties are conjugated thereto, one at a 5′ end and one at a 3′ end ofthe oligonucleotide. Oligonucleotides or analogs having from 2 to 3quenching moieties may be synthesized using conventional linkage andsynthetic chemistries, for example, as disclosed in the references citedherein.

Oligonucleotides or analogs may be provided as a single species or theymay be provided as mixtures of a plurality of oligonucleotides oranalogs with different sequences, and therefore, different bindingspecificities. In some embodiments, oligonucleotides or analogs arerandom sequence polymers; that is, they are provided as mixtures ofevery possible sequence of a given length. For example, sucholigonucleotides or analogs may be represented by the formulas, “NNNNNN”for 6-mers, or “NNNNNNNN” for 8-mers, wherein N may be A, C, G or T, oran analog thereof.

“Analogs” in reference to oligonucleotides means an oligonucleotide thatcontains one or more nucleotide analogs. As described in the definitionsection, a “nucleotide analog” is a nucleotide that may have a modifiedlinkage moiety, sugar moiety or base moiety. Exemplary oligonucleotideanalogs that may be used with the invention include, but are not limitedto, peptide nucleic acids (PNAs), locked nucleic acids(LNAs)(2′-O-methyl RNA), phosphorothioate oligonucleotides, bridgednucleic acids (BNAs), or the like.

In some embodiments, oligonucleotide binding moieties comprise universalbases; that is, they contain one or more nucleotide analogs that canreplace any of the four natural nucleotides without destabilizingbase-pair interactions. Nucleotide analogs having universal baseproperties are described in Loakes, Nucleic Acids Research, 29(12):2437-2447 (2001), which is incorporated herein by reference. In someembodiments, oligonucleotide binding moieties comprise 2′-deoxyinosine,7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine, 3-nitropyrrolenucleotides, 5-nitroindole nucleotides, or the like.

In some embodiments, quenching agents may comprise a combination of twoor more compounds that act together to quench undesired fluorescentsignals of a single stranded labeled polynucleotide. For example, aquenching agent may comprise an oligonucleotide (e.g., polydeoxyinosine)that may form a duplex with the labeled polynucleotide and separately adouble stranded intercalator that is a quencher. Thus, whenever thepolydeoxyinosine binds to a labeled polynucleotide, the quenchingintercalator binds to the resulting duplex and quenches fluorescentsignals from the polynucleotide.

Any synthetic dye that can detectably quench fluorescent signals of thefluorescent labels of a labeled polynucleotide is an acceptablequenching moiety for the purposes of the invention. Specifically, asused in the invention, the quenching moieties possess an absorption bandthat exhibits at least some spectral overlap with an emission band ofthe fluorescent labels on a labeled polynucleotide. This overlap mayoccur with emission of the fluorescent label (donor) occurring at alower or even higher wavelength emission maximum than the maximalabsorbance wavelength of the quenching moiety (acceptor), provided thatsufficient spectral overlap exists. Energy transfer may also occurthrough transfer of emission of the donor to higher electronic states ofthe acceptor. One of ordinary skill in the art determines the utility ofa given quenching moiety by examination of that dye's excitation bandswith respect to the emission spectrum of the fluorescent labels beingused.

Typically, fluorescence quenching in the invention occurs throughFluorescence Resonance Energy Transfer (FRET or through the formation ofcharge transfer complexes) between a fluorescent label and a quenchingmoiety of the invention. The spectral and electronic properties of thedonor and acceptor compounds have a strong effect on the degree ofenergy transfer observed, as does the separation distance between thefluorescent labels on the labeled polynucleotide and the quenchingmoiety. As the separation distance increases, the degree of fluorescencequenching decreases.

A quenching moiety may be optionally fluorescent, provided that themaximal emission wavelength of the dye is well separated from themaximal emission wavelength of the fluorescent labels when bound tolabeled polynucleotides. Preferably, however, the quenching moiety isonly dimly fluorescent, or is substantially non-fluorescent, whencovalently conjugated to a oligonucleotide or analog. Substantiallynon-fluorescent, as used herein, indicates that the fluorescenceefficiency of the quenching moiety in an assay solution as described forany of the methods herein is less than or equal to 5 percent, preferablyless than or equal to 1 percent. In other embodiments, the covalentlybound quenching moiety exhibits a quantum yield of less than about 0.1,more preferably less than about 0.01. In some embodiments, thefluorescence of fluorescent labels associated with a quenchingoligonucleotide of the invention is quenched more than 50% relative tothe same oligonucleotide associated with the same fluorescent labels inthe absence of the covalently bound quenching moiety. In anotherembodiment, the fluorescent labels are quenched more than 90% relativeto the unlabeled oligonucleotide. In yet another embodiment, the nucleicacid stains are quenched more than 95% relative to the unlabeledoligonucleotide.

In some embodiments, a quenching moiety may be a pyrene, an anthracene,a naphthalene, an acridine, a stilbene, an indole or benzindole, anoxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine,a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a coumarin (includinghydroxycoumarins and aminocoumarins and fluorinated and sulfonatedderivatives thereof (as described in U.S. Pat. No. 5,830,912 to Gee etal. (1998) and U.S. Pat. No. 5,696,157 to Wang et al. (1997),incorporated by reference), a polyazaindacene (e.g. U.S. Pat. No.4,774,339 to Haugland, et al. (1988); U.S. Pat. No. 5,187,288 to Kang,et al. (1993); U.S. Pat. No. 5,248,782 to Haugland, et al. (1993); U.S.Pat. No. 5,274,113 to Kang, et al. (1993); U.S. Pat. No. 5,433,896 toKang, et al. (1995); U.S. Pat. No. 6,005,113 to Wu et al. (1999), allincorporated by reference), a xanthene, an oxazine or a benzoxazine, acarbazine (U.S. Pat. No. 4,810,636 to Corey (1989), incorporated byreference), or a phenalenone or benzphenalenone (U.S. Pat. No. 4,812,409Babb et al. (1989), incorporated by reference).

In other embodiments, quenching moieties that are substantiallynon-fluorescent dyes include in particular azo dyes (such as DABCYL orDAB SYL dyes and their structural analogs), triarylmethane dyes such asmalachite green or phenol red, 4′,5z-diether substituted fluoresceins(U.S. Pat. No. 4,318,846 (1982)), or asymmetric cyanine dye quenchers(PCT Int. App. WO 99 37,717 (1999)).

In embodiments where the quenching moiety is a xanthene, the syntheticdye is optionally a fluorescein, a rhodol (U.S. Pat. No. 5,227,487 toHaugland, et al. (1993), incorporated by reference), or a rhodamine. Asused herein, fluorescein includes benzo- or dibenzofluoresceins,seminaphthofluoresceins, or naphthofluoresceins. Similarly, as usedherein rhodol includes seminaphthorhodafluors (U.S. Pat. No. 4,945,171to Haugland, et al. (1990), incorporated by reference). Xanthenesinclude fluorinated derivatives of xanthene dyes (Int. Publ. No. WO97/39064, Molecular Probes, Inc. (1997), incorporated by reference), andsulfonated derivatives of xanthene dyes (Int. Publ. No. WO 99/15517,Molecular Probes, Inc. (1999), incorporated by reference). As usedherein, oxazines include resorufms, aminooxazinones, diaminooxazines,and their benzo-substituted analogs.

In further embodiments, the quenching moiety is an substantiallynonfluorescent derivative of 3- and/or 6-amino xanthene that issubstituted at one or more amino nitrogen atoms by an aromatic orheteroaromatic ring system, e.g. as described in U.S. Pat. No.6,399,392, which is incorporated herein by reference. These quenchingdyes typically have absorption maxima above 530 nm, have little or noobservable fluorescence and efficiently quench a broad spectrum ofluminescent emission, such as is emitted by chemilumiphores, phosphors,or fluorophores. In one embodiment, the quenching dye is a substitutedrhodamine. In another embodiment, the quenching compound is asubstituted rhodol.

In still other embodiments, a quenching moiety may comprise one or morenon-fluorescent quenchers known as Black Hole Quenchers™ compounds(BHQs) described in the following patents, which are incorporated hereinby reference: U.S. Pat. Nos. 7,019,129; 7,109,312; 7,582,432; 8,410,025;8,440,399; 8,633,307; 8,946,404; 9,018,369; or 9,139,610.

Additional quenching moieties are disclosed in the following, which areincorporated herein by reference: U.S. Pat. Nos. 6,699,975; 6,790,945;and 8,114,979.

EXAMPLE Translocation of Target Polynucleotide in an Optically-BasedNanopore Sequencing Method

In this example, the invention is used in conjunction with an exemplaryoptically-based nanopore sequencing method. In the exemplaryoptically-based nanopore sequencing method, nucleotides of targetpolynucleotides are labeled with fluorescent labels that are capable ofat least three states: (i) A quenched state wherein fluorescence of anattached fluorescent label is quenched by a fluorescent label on animmediately adjacent nucleotide; for example, a fluorescent labelattached to a polynucleotide is quenched when the labeled polynucleotideis free in an aqueous solution. (ii) A sterically constrained statewherein a labeled polynucleotide is translocating through a nanoporesuch that the free-solution movements or alignments of an attachedfluorescent label is disrupted or limited so that there is little or nodetectable signal generated from the fluorescent label. (iii) Atransition state wherein a fluorescent label attached to apolynucleotide transitions from the sterically constrained state to thequenched state as the fluorescent label exits the nanopore (during a“transition interval”) while the polynucleotide translocates through thenanopore. A nucleotide sequence of a polynucleotide is determined byrecording signals generated by attached fluorescent labels as they exita nanopore one at a time as a polynucleotide translocates the nanopore.Upon exit, each attached fluorescent label transitions during atransition interval from a constrained state in the nanopore to aquenched state on the polynucleotide in free solution. During thistransition interval the fluorescent label is capable of emitting adetectable fluorescent signal indicative of the nucleotide it isattached to.

In some embodiments, the invention may be used with such a nanoporesequencing method using the following steps: (a) extending a primerhaving a 5′ non-complementary tail on a template in a reaction mixtureto produce a double stranded product comprising an extended strand andthe 5′ non-complementary tail as a single stranded overhang; (b)providing a nanopore (or an array of nanopores) that separates andprovides fluid communication between a first chamber and a secondchamber, wherein the nanopore is capable of passing a single strandednucleic acid but not a double stranded nucleic acid; (c) disposing thedouble stranded product in the first chamber; (d) capturing the 5′non-complementary tail of the isolated double stranded product by thenanopore by applying an electrical field across the nanopore; (e)translocating a polymer analyte through a nanopore having a bore and anexit, the polymer analyte comprising a sequence of monomers, whereinsubstantially each monomer is labeled with a fluorescent label such thatfluorescent labels of adjacent monomers are in a quenched state byself-quenching one another outside of the nanopore and fluorescentlabels are in a sterically constrained state and incapable of generatinga detectable fluorescent signal inside of the nanopore; (f) excitingeach fluorescent label at the exit of the nanopore as it transitionsfrom a sterically constrained state to a quenched state so that afluorescent signal is generated which is indicative of the monomer towhich it is attached; (g) detecting the fluorescent signal to identifythe monomer. As used herein, “substantially every”, “substantially all”,or like terms, in reference to labeling monomers, particularlynucleotides, acknowledges that chemical labeling procedures may notresult in complete labeling of every monomer; to the extent practicable,the terms comprehend that labeling reactions in connection with theinvention are continued to completion; in some embodiments, suchcompleted labeling reactions include labeling at least fifty percent ofthe monomers; in other embodiments, such labeling reactions includelabeling at least eighty percent of the monomers; in other embodiments,such labeling reactions include labeling at least ninety-five percent ofthe monomers; in other embodiments, such labeling reactions includelabeling at least ninety-nine percent of the monomers.

In some embodiments of the above method, fluorescent labels are membersof a FRET pair. A FRET pair generally is one or more FRET donors and oneor more FRET acceptors where each donor is capable of a FRET reactionwith each acceptor. In one aspect, this means that the donors of theFRET pair have an emission spectrum that substantially overlaps theabsorption spectrum of the acceptors. In another aspect, the transitiondipole of the donor and the acceptor have to be aligned in a way thatallows efficient energy transfer. In some aspects, the invention in partis based on the discovery and appreciation of a fluorescence,particularly, FRET suppressing property of nanopores and the applicationof this property to enable detection of labeled polymers translocatingthrough a nanopore. It is believed, although the invention is notintended to be limited thereby, that a nanopore may be selected with abore dimensioned so that a FRET pair label cannot orient to engage in aFRET interaction while translocating through the nanopore. The dipolesof the labels of the polynucleoide in the bore of the nanopore areconstrained in their rotational freedom based on the limited diameter ofthe nanopore. This reduction in dipole alignment with the alignment ofthe corresponding FRET pair attached to the nanopore limits the FRETefficiency dramatically. Labeled polynucleotides can engage in a FRETinteraction after exiting the nanopore at which point the FRET acceptoror donor on the polymer (e.g. polynucleotide) regains rotational freedomwhich allows for a FRET event.

Definitions

“FRET” or “Förster, or fluorescence, resonant energy transfer” means anon-radiative dipole-dipole energy transfer mechanism from an exciteddonor fluorophore to an acceptor fluorophore in a ground state. The rateof energy transfer in a FRET interaction depends on the extent ofspectral overlap of the emission spectrum of the donor with theabsorption spectrum of the acceptor, the quantum yield of the donor, therelative orientation of the donor and acceptor transition dipoles, andthe distance between the donor and acceptor molecules, Lakowitz,Principles of Fluorescence Spectroscopy, Third Edition (Springer, 2006).FRET interactions of particular interest are those which result aportion of the energy being transferred to an acceptor, in turn, beingemitted by the acceptor as a photon, with a frequency lower than that ofthe light exciting its donor (i.e. a “FRET signal”). “FRET distance”means a distance between a FRET donor and a FRET acceptor over which aFRET interaction can take place and a detectable FRET signal produced bythe FRET acceptor.

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., fluorescentlabels, such as mutually quenching fluorescent labels, fluorescent labellinking agents, enzymes, etc. in the appropriate containers) and/orsupporting materials (e.g., buffers, written instructions for performingthe assay etc.) from one location to another. For example, kits includeone or more enclosures (e.g., boxes) containing the relevant reactionreagents and/or supporting materials. Such contents may be delivered tothe intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a second ormore containers contain mutually quenching fluorescent labels.

“Nanopore” means any opening positioned in a substrate that allows thepassage of analytes through the substrate in a predetermined ordiscernable order, or in the case of polymer analytes, passage of theirmonomeric units through the substrate in a pretermined or discernibleorder. In the latter case, a predetermined or discernible order may bethe primary sequence of monomeric units in the polymer. Examples ofnanopores include proteinaceous or protein based nanopores, synthetic orsolid state nanopores, and hybrid nanopores comprising a solid statenanopore having a protein nanopore embedded therein. A nanopore may havean inner diameter of 1-10 nm or 1-5 nm or 1-3 nm. Examples of proteinnanopores include but are not limited to, alpha-hemolysin,voltage-dependent mitochondrial porin (VDAC), OmpF, OmpC, MspA and LamB(maltoporin), e.g. disclosed in Rhee, M. et al., Trends inBiotechnology, 25(4) (2007): 174-181; Bayley et al (cited above);Gundlach et al, U.S. patent publication 2012/0055792; and the like,which are incorporated herein by reference. Any protein pore that allowsthe translocation of single nucleic acid molecules may be employed. Ananopore protein may be labeled at a specific site on the exterior ofthe pore, or at a specific site on the exterior of one or more monomerunits making up the pore forming protein. Pore proteins are chosen froma group of proteins such as, but not limited to, alpha-hemolysin, MspA,voltage-dependent mitochondrial porin (VDAC), Anthrax porin, OmpF, OmpCand LamB (maltoporin). Integration of the pore protein into the solidstate hole is accomplished by attaching a charged polymer to the poreprotein. After applying an electric field the charged complex iselectrophoretically pulled into the solid state hole. A syntheticnanopore, or solid-state nanopore, may be created in various forms ofsolid substrates, examples of which include but are not limited tosilicones (e.g. Si3N4, SiO2), metals, metal oxides (e.g. Al2O3)plastics, glass, semiconductor material, and combinations thereof. Asynthetic nanopore may be more stable than a biological protein porepositioned in a lipid bilayer membrane. A synthetic nanopore may also becreated by using a carbon nanotube embedded in a suitable substrate suchas but not limited to polymerized epoxy. Carbon nanotubes can haveuniform and well-defined chemical and structural properties. Varioussized carbon nanotubes can be obtained, ranging from one to hundreds ofnanometers. The surface charge of a carbon nanotube is known to be aboutzero, and as a result, electrophoretic transport of a nucleic acidthrough the nanopore becomes simple and predictable (Ito, T. et al.,Chem. Commun. 12 (2003): 1482-83). The substrate surface of a syntheticnanopore may be chemically modified to allow for covalent attachment ofthe protein pore or to render the surface properties suitable foroptical nanopore sequencing. Such surface modifications can be covalentor non-covalent. Most covalent modification include an organosilanedeposition for which the most common protocols are described:1)Deposition from aqueous alcohol. This is the most facile method forpreparing silylated surfaces. A 95% ethanol-5% water solution isadjusted to pH 4.5-5.5 with acetic acid. Silane is added with stirringto yield a 2% final concentration. After hydrolysis and silanol groupformation the substrate is added for 2-5 min. After rinsed free ofexcess materials by dipping briefly in ethanol. Cure of the silane layeris for 5-10 min at 110 degrees Celsius. 2) Vapor Phase Deposition.Silanes can be applied to substrates under dry aprotic conditions bychemical vapor deposition methods. These methods favor monolayerdeposition. In closed chamber designs, substrates are heated tosufficient temperature to achieve 5 mm vapor pressure. Alternatively,vacuum can be applied until silane evaporation is observed. 3) Spin-ondeposition. Spin-on applications can be made under hydrolytic conditionswhich favor maximum functionalization and polylayer deposition or dryconditions which favor monolayer deposition. In some embodiments, singlenanopores are employed with methods of the invention. In otherembodiments, a plurality of nanopores are employed. In some of thelatter embodiments, a plurality of nanopores is employed as an array ofnanopores, usually disposed in a planar substrate, such as a solid phasemembrane. Nanopores of a nanopore array may be spaced regularly, forexample, in a rectilinear pattern, or may be spaced randomly. In apreferred embodiment, nanopores are spaced regularly in a rectilinearpattern in a planar solid phase substrate.

“Nanostructure” (used interchangeably with “nanoscale structure” and“nanoscale feature”) means a structure that has at least one dimensionwithin a range of a few nanometers to several hundred nanometers, forexample, from 1 to 1000 nanometers. In some applications, such range isfrom 2 to 500 nanometers; in other applications, such range is from 3 to500 nanometers. The shape and geometry of nanostructures may vary widelyand include, but are not limited to, nanopores, nanowells,nanoparticles, and any other convenient shapes particularly suitable forcarrying out sequences of reactions. In some embodiments, nanostructuresmay be protein nanopores operationally associated with a solid phasemembrane. Some nanostructures, such as, nanopores and nanowells, may beformed in a larger common substrate, such as a solid phase membrane, orother solid, to form arrays of nanopores or nanowells. Nanostructures ofparticular interest are those capable of supporting or containing achemical, physical (e.g. FRET), enzymatic and/or binding reaction or asequence of such reactions. In some embodiments, a nanostructure, suchas a nanowell, encloses a volume that is less than one nanoliter (10×-9liter), less than one picoliter, or less than one femtoliter. In otherembodiments, each of the individual nanowells provides a volume that isless than 1000 zeptoliters, 100 zeptoliters, 80 zeptoliters, or lessthan 50 zeptoliters, or less than 1 zeptoliter, or even less than 100yactoliters. In some embodiments, nanowells comprise zero modewaveguides.

“Peptide,” “peptide fragment,” “polypeptide,” “oligopeptide,” or“fragment” in reference to a peptide are used synonymously herein andrefer to a compound made up of a single unbranched chain of amino acidresidues linked by peptide bonds. Amino acids in a peptide orpolypeptide may be derivatized with various moieties, including but notlimited to, polyethylene glycol, dyes, biotin, haptens, or likemoieties. The number of amino acid residues in a protein or polypeptideor peptide may vary widely; however, in some embodiments, protein orpolypeptides or peptides referred to herein may have 2 from to 70 aminoacid residues; and in other embodiments, they may have from 2 to 50amino acid residues. In other embodiments, proteins or polypeptides orpeptides referred to herein may have from a few tens of amino acidresidues, e.g. 20, to up to a thousand or more amino acid residues, e.g.1200. In still other embodiments, proteins, polypeptides, peptides, orfragments thereof, may have from 10 to 1000 amino acid residues; or theymay have from 20 to 500 amino acid residues; or they may have from 20 to200 amino acid residues.

“Polymer” means a plurality of monomers connected into a linear chain.Usually, polymers comprise more than one type of monomer, for example,as a polynucleotide comprising A's, C's, G's and T's, or a polypeptidecomprising more than one kind of amino acid. Monomers may includewithout limitation nucleosides and derivatives or analogs thereof andamino acids and derivatives and analogs thereof. In some embodiments,polymers are polynucleotides, whereby nucleoside monomers are connectedby phosphodiester linkages, or analogs thereof.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like. Such monomers and their internucleosidic linkagesmay be naturally occurring or may be analogs thereof, e.g. naturallyoccurring or non-naturally occurring analogs. Non-naturally occurringanalogs may include PNAs, phosphorothioate internucleosidic linkages,bases containing linking groups permitting the attachment of labels,such as fluorophores, or haptens, and the like. Whenever the use of anoligonucleotide or polynucleotide requires enzymatic processing, such asextension by a polymerase, ligation by a ligase, or the like, one ofordinary skill would understand that oligonucleotides or polynucleotidesin those instances would not contain certain analogs of internucleosidiclinkages, sugar moieties, or bases at any or some positions.Polynucleotides typically range in size from a few monomeric units, e.g.5-40, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. Whenever a polynucleotide oroligonucleotide is represented by a sequence of letters (upper or lowercase), such as “ATGCCTG,” it will be understood that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesthymidine, “I” denotes deoxyinosine, “U” denotes uridine, unlessotherwise indicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references. Likewise, theoligonucleotide and polynucleotide may refer to either a single strandedform or a double stranded form (i.e. duplexes of an oligonucleotide orpolynucleotide and its respective complement). It will be clear to oneof ordinary skill which form or whether both forms are intended from thecontext of the terms usage.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring HarborPress, New York, 2003).

“Sequence determination”, “sequencing” or “determining a nucleotidesequence” or like terms in reference to polynucleotides includesdetermination of partial as well as full sequence information of thepolynucleotide. That is, the terms include sequences of subsets of thefull set of four natural nucleotides, A, C, G and T, such as, forexample, a sequence of just A's and C's of a target polynucleotide. Thatis, the terms include the determination of the identities, ordering, andlocations of one, two, three or all of the four types of nucleotideswithin a target polynucleotide. In some embodiments, the terms includethe determination of the identities, ordering, and locations of two,three or all of the four types of nucleotides within a targetpolynucleotide. In some embodiments sequence determination may beaccomplished by identifying the ordering and locations of a single typeof nucleotide, e.g. cytosines, within the target polynucleotide “catcgc. . . ” so that its sequence is represented as a binary code, e.g.“100101 . . . ” representing “c-(not c)(not c)c-(not c)-c . . . ” andthe like. In some embodiments, the terms may also include subsequencesof a target polynucleotide that serve as a fingerprint for the targetpolynucleotide; that is, subsequences that uniquely identify a targetpolynucleotide, or a class of target polynucleotides, within a set ofpolynucleotides, e.g. all different RNA sequences expressed by a cell.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations described herein.Further, the scope of the disclosure fully encompasses other variationsthat may become obvious to those skilled in the art in view of thisdisclosure. The scope of the present invention is limited only by theappended claims.

What is claimed is:
 1. A method of determining characteristics ofpolymers, the method comprising: providing a nanopore array comprising asolid phase membrane and an opaque layer co-extensive therewith, thenanopore array comprising a plurality of apertures and separating afirst chamber and a second chamber, wherein each aperture provides fluidcommunication between the first chamber and the second chamber and has asignal generation region and wherein the opaque layer substantiallyprevents light from passing through the nanopore array; translocatingpolymers from the first chamber to the second chamber through theapertures, each polymer having one or more optical labels attachedthereto capable of generating an optical signal having at least a firstwavelength indicative of a characteristic of the polymer; exciting withan excitation beam having a second wavelength the optical labels of thepolymers as they translocate through the signal generation regions ofthe apertures, wherein the optical labels in the detection regionsgenerate optical signals whose first wavelength is different than thesecond wavelength; detecting optical signals from the optical labels inthe signal generation regions to determine the characteristics of thepolymers.
 2. The method of claim 1 wherein said opaque layer is a metallayer.
 3. The method of claim 2 wherein said metal layer comprises ametal selected from the group consisting of Al, Au, Ag and Cu.
 4. Themethod of claim 1 wherein said polymers are polynucleotides and whereinsaid characteristic is a nucleotide sequence thereof.
 5. The method ofclaim 4 wherein said optical labels are fluorescent labels and whereinsaid optical signals are fluorescent signals.
 6. The method of claim 5wherein said signal generation region of each of said apertures extendsfrom a surface of said opaque layer closest to said second chamber intosaid second chamber.
 7. The method of claim 6 wherein different kinds ofnucleotides of said polynucleotide are labeled with differentfluorescent labels that generate distinguishable fluorescent signals andwherein each of said apertures constrains nucleotides of apolynucleotide to move single file through said signal generationregion.
 8. The method of claim 7 further including a step of quenchingsaid fluorescent signals from excited fluorescent labels outside of saidsignal generation region using a non-fluorescent quenching agent.
 9. Themethod of claim 8 wherein said quenching agent binds to saidpolynucleotides.
 10. The method of claim 9 wherein said quenching agentis disposed in said second chamber.
 11. The method of claim 5 furtherincluding a step of quenching said fluorescent signals from excitedfluorescent labels outside of said signal generation region by selectingsaid fluorescent labels to be mutually self-quenching.
 12. The method ofclaim 1 wherein said excitation beam is directed to said nanopore arraythrough said second chamber so that said opaque layer substantiallyprevents excitation of optical labels in said first chamber.
 13. Themethod of claim 1 wherein each of said apertures of said nanopore arraycomprises a protein nanopore immobilized therein.
 14. The method ofclaim 1 wherein said optical labels are acceptor labels and wherein saidexcitation beam excites a donor label at each of said apertures whichexcites acceptor labels as they translocate through said signalgeneration region.
 15. A method of determining sequences ofpolynucleotides, the method comprising: providing a nanopore arraycomprising a solid phase membrane and an opaque layer co-extensivetherewith, the nanopore array comprising a plurality of apertures andseparating a first chamber and a second chamber, wherein each apertureprovides fluid communication between the first chamber and the secondchamber and has a signal generation region and wherein the opaque layersubstantially prevents light from passing through the nanopore array;translocating polynucleotides from the first chamber to the secondchamber through the apertures, wherein different kinds of nucleotides ofthe polynucleotides are labeled with different fluorescent labels thatgenerate distinguishable fluorescent signals and wherein each of saidapertures constrains nucleotides of a polynucleotide to move single filethrough the signal generation region; exciting with an excitation beamthe fluorescent labels of the polynucleotides as they translocatethrough the signal generation regions of the apertures; detectingfluorescent signals from the fluorescent labels in the signal generationregions to determine the characteristics of the polymers; anddetermining a sequence of nucleotides from the fluorescent signalsdetected at the signal generation region of each aperture.
 16. Themethod of claim 15 wherein said opaque layer is a metal layer.
 17. Themethod of claim 16 wherein said metal layer comprises an aluminum layeror a gold layer.
 18. The method of claim 16 wherein said signalgeneration region of each of said apertures extends from a surface ofsaid metal layer closest to said second chamber into said secondchamber.
 19. The method of claim 16 wherein said excitation beam isdirected to said nanopore array through said second chamber so that saidmetal layer substantially prevents excitation of optical labels in saidfirst chamber.
 20. The method of claim 16 further including a step ofquenching said fluorescent signals from excited fluorescent labelsoutside of said signal generation region using a non-fluorescentquenching agent.
 21. The method of claim 20 wherein said quenching agentbinds to said polynucleotides.
 22. The method of claim 21 wherein saidquenching agent is disposed in said second chamber.
 23. The method ofclaim 16 further including a step of quenching said fluorescent signalsfrom excited fluorescent labels outside of said signal generation regionby selecting said fluorescent labels to be mutually self-quenching. 24.The method of claim 16 wherein said steps of exciting and detecting areimplemented with an epi-illumination system.
 25. The method of claim 16wherein each of said apertures of said nanopore array comprises aprotein nanopore immobilized therein.
 26. The method of claim 25 whereineach of said protein nanopores are immobilized in a lipid bilayerdisposed across said apertures.
 27. The method of claim 16 wherein saidfluorescent labels are acceptor labels and wherein said excitation beamexcites a donor label at each of said apertures which donor labelsexcite the acceptor labels as they translocate through said signalgeneration region.